Cyclic GMP dependent protien kinase as a chemotherapeutic target for antiprotozoal agents

ABSTRACT

Protozoal cyclic GMP dependent protein kinases have been isolated and cloned. These enzymes may be used in screening assays to identify potential antiprotozoal agents.

SUMMARY OF THE INVENTION

[0001] The present invention relates to a novel protein kinase which maybe used as a chemotherapeutic target for antiprotozoal agents. Theinvention further concerns a method for identifying potentialantiprotozoal agents using the novel kinase, as well as a method fortreating protozoal infections using a substance that inhibits the actionof said kinase.

BACKGROUND OF THE INVENTION

[0002] Parasitic protozoa are responsible for a wide variety ofinfections in man and animals. Many of the diseases are life threateningto the host, and in animal husbandry, can cause considerable economicloss. For example, malaria remains a significant health threat to humansdespite massive international attempts to eradicate the disease;trypanosomiasis such as Chagas disease caused by Trypanosoma cruzi andAfrican sleeping sickness caused by T. brucei are not uncommon in SouthAmerica and Africa, respectively; and opportunistic infections inimmuno-compromised hosts caused by Pneumocystis carinii, Toxoplasmagondii, Cryptosporidium sp. are becoming increasingly significant in thedeveloped countries.

[0003] Coccidiosis, a widespread disease of domesticated animals, iscaused by protozoal infection. In the poultry industry, coccidiosis isresponsible for high levels of morbidity and mortality in the birdpopulation and may result in extreme economic losses. The infectiousagents are protozoa of the genus Eimeria. Some of the most significantavian Eimeria species include E. tenella, E. acervulina, E. necatrix, E.brunetti and E. maxima.

[0004] In some protozoal diseases, such as Chagas disease, there is nosatisfactory treatment; in others, drug-resistant strains of theprotozoa may develop. Accordingly, there exists a continued need toidentify new and effective anti-protozoal drugs. However, antiparasiticdrug discovery has been, for the most part, a random and laboriousprocess through biological screening of natural products and syntheticcompounds against a panel of parasites. This process can be greatlyfacilitated and made more specific if a biochemical target ofantiprotozoal drugs can be identified, and incorporated into thescreening process.

[0005] cGMP dependent protein kinases (PKG) catalyze the phosphorylationof specific protein substrates. In the absence of cGMP the activity ofthese enzymes is very low. In mammalian cells there are two types ofPKG, a soluble (PKG1) and a membrane bound form (PKG2). Multiple splicevariants of the soluble protein have been identified. PKGs are known tocontrol many cellular processes in higher animals. Mammalian PKG1 ismost abundant in smooth muscle, platelets and cerebellum. Targeteddisruption of PKG1 in mice generated phenotypes clearly associated withsmooth muscle, namely severe intestinal and vascular dysfunctions. PKG2expression is highest in the small intestine, several regions of thebrain (particularly the hypothalamus) and lung. Transgenic mice lackingPKG2 display a dwarfed phenotype caused by defects in ossification atthe growth plates and also have intestinal secretion dysfunctions. PKGshave also been identified in Dictyostelium, Paramecium, Tetrahymena andAscaris.

DETAILED DESCRIPTION OF THE INVENTION

[0006] In one aspect the present invention provides a novel protozoalcGMP dependent protein kinase (PKG) and a polynucleotide sequenceencoding the PKG polypeptide. In one embodiment, the invention providesa PKG purified fraction of Eimeria tenella consisting essentially of theamino acid sequence of SEQ ID NO: 11, and the polynucleotide sequenceencoding the polypeptide of SEQ ID NO: 11. More specifically, thepolynucleotide sequences is as set forth in SEQ ID NO: 10. In anotherembodiment, the invention provides a substantially pure PKG ofToxoplasma gondii consisting essentially of the amino acid sequence ofSEQ ID NO: 13, and the polynucleotide sequence encoding the polypeptideof SEQ ID NO: 13. More specifically, the polynucleotide sequences is asset forth in SEQ ID NO: 12.

[0007] In another aspect the present invention provides a method foridentifying compounds having antiprotozoal activity comprising:

[0008] (a) contacting protozoal PKG with, (i) a known amount of alabeled compound that interacts with a PKG and, (ii) a known dilution ofa test compound or a natural product extract; and

[0009] (b) quantitating the percent inhibition of interaction of saidlabeled compound induced by said test compound.

[0010] In another aspect the present invention provides a method foridentifying compounds having antiprotozoal activity comprising:

[0011] (a) contacting an intact host or protozoal cell with a testcompound or a natural product extract;

[0012] (b) disrupting said cell to obtain a biochemical fractionpossessing PKG catalytic activity; and

[0013] (c) determining the level of PKG activity in said biochemicalfraction.

[0014] The methods of the invention provides a facile and specific assayto screen compounds as potential antiprotozoal drugs.

[0015] Polypeptides

[0016] The novel PKG of the present invention is of protozoal origin; inparticular the enzyme is present in, but not restricted to, protozoa ofthe apicomplexan family, and more specifically in Eimeria sp. The nativeEimeria teneila PKG of the present invention is a protein of about 120kDa, and having about 1,003 amino acids. The native PKG from Toxoplasmagondii is approximately 115 kDa and having about 994 amino acids. ThePKG of the present invention includes a crude extract of the solubleprotein, a PKG purified fraction isolated from a protozoan parasite(native enzyme), affinity purified native PKG (purified from a solubleextract using cGMP, substrate based peptides, inhibitor based moleculesor antibodies) as well as a PKG produced by recombinant DNA technology(recombinant expressed enzyme). The term “PKG purified fraction” as usedherein, refers to a PKG polypeptide which is free of most otherproteins, lipids, carbohydrates, nucleic acids, or other materials withwhich it is naturally associated. One skilled in the art can purify PKGusing standard techniques for protein purification. The PKG purifiedfraction will yield a major band on a reducing polyacrylamide gel. Thesequence of the polypeptide can be determined by amino acid sequencing.

[0017] The protozoal PKGs of the present invention include a polypeptideof SEQ ID NO: 11, and a polypeptide of SEQ ID NO: 13, as well asfunctional polypeptides and fragments thereof. As used herein, the term“functional polypeptides and fragments” refers to a polypeptide whichpossesses PKG activity. Minor modifications of the PKG primary aminoacid sequence may result in proteins which have substantially equivalentactivity as compared to the PKG polypeptide described herein. Suchmodifications may be deliberate, as by site-directed mutagenesis, or maybe spontaneous. All of the polypeptides produced by these modificationsare included herein as long as the enzymatic activity of PKG is present.Further, deletion of one or more amino acids can also result in amodification of the structure of the resultant molecule withoutsignificantly altering its kinase activity. This can lead to thedevelopment of a smaller active molecule which may have broader utility.For example, it is possible to remove amino or carboxyl terminal aminoacids which may not be required for kinase activity. Smaller peptidescontaining the biological activity of PKG are included in the invention.

[0018] The PKG polypeptide of the invention also includes conservativevariations of the polypeptide sequence which do not substantially alterthe biological activity of the protein. The term “conservativevariation” as used herein denotes the replacement of an amino acidresidue by another, biologically similar residue. Examples ofconservative variations include the substitution of one hydrophobicresidue such as isoleucine, valine or leucine for another, or thesubstitution of one polar residue for another, such as the substitutionof arginine for lysine, glutamic for aspattic acids, or glutamine forasparagine, and the like. The term “conservative variation” alsoincludes the use of a substituted amino acid in place of anunsubstituted parent amino acid provided that the activity of the enzymeis not substantially altered.

[0019] Polynucleotides

[0020] The invention also provides isolated polynucleotide sequencesconsisting essentially of a polynucleotide sequence encoding apolypeptide having the amino acid sequence of SEQ ID NO: 11 or SEQ IDNO: 13. As used herein, “polynucleotide” refers to apolydeoxyribonucleotides or polyribonucleotides, in the form of aseparate fragment or a larger construct, and includes DNA, cDNA and RNAsequences which encode protozoal PKG. The term “isolated” as used hereinincludes polynucleotides substantially free of other nucleic acids,proteins, lipids, carbohydrates or other materials with which it isnaturally associated. It is understood that all polynucleotides encodingall or a portion of protozoal PKG are also included herein, as long asthey encode a polypeptide with PKG kinase activity. Such polynucleotidesinclude naturally occurring, synthetic, and intentionally manipulatedpolynucleotides. For example, PKG polynucleotide may be subjected tosite-directed mutagenesis.

[0021] It is known that there is a substantial amount of redundancy inthe various codons which code for specific amino acids. There are 20natural amino acids, most of which are specified by more than one codon.Therefore, this invention is also directed to those DNA sequences whichcontain alternative codons which code for the eventual translation ofthe identical amino acid. For purposes of this specification, a sequencebearing one or more replaced codons will be defined as a degeneratevariation. Therefore, the present invention discloses codon redundancywhich may result in differing DNA molecules expressing an identicalprotein.

[0022] Specifically disclosed herein is a cDNA sequence 4283 base pair(bp) in length (SEQ. ID NO: 10) containing the predicted coding regionfor E. tenella PKG. The cDNA includes an open reading frame of 3009 basepairs encoding a protein of about 1003 amino acids, having a deducedmolecular weight of about 113 kDa. Also specificallydisclosed herein isa cDNA sequence 3898 base pair (bp) in length (SEQ. ID NO: 12)containing the predicted coding region for T. gondii PKG. The cDNAincludes an open reading frame of 2982 base pairs encoding a protein ofabout 994 amino acids, having a deduced molecular weight of about 112kDa. The polynucleotides encoding PKG include the nucleotide sequencesSEQ ID NO: 10 and SEQ ID NO: 12, as well as nucleic acid sequencescomplementary to that sequence. A complementary sequence may include anantisense nucleotide. When the sequence is RNA, the deoxynucleotides A,G, C, and T are replaced by ribonucleotides A, G, C, and U,respectively. Also included in the invention are fragments of theabove-described nucleic acid sequences that are at least 15 bases inlength, which is sufficient to permit the fragment to selectivelyhybridize to DNA that encodes the proteins of the present inventionincluding PKG of SEQ ID NO: 11 and SEQ ID NO: 13.

[0023] Any of a variety of procedures may be used to clone PKG. Thesemethods include, but are not limited to,

[0024] (1) a RACE PCR cloning technique (Frohman, et al., 1988, Proc.Natl. Acad. Sci.85: 8998-9002). 5′ and/or 3′ RACE may be performed togenerate a full length cDNA sequence. This strategy involves usinggene-specific oligonucleotide primers for PCR amplification of PKG cDNA.These gene-specific primers are designed through identification of anexpressed sequence tag (EST) nucleotide sequence which has beenidentified by searching any number of publicly available nucleic acidand protein databases;

[0025] (2) direct functional expression of the PKG cDNA following theconstruction of a PKG-containing cDNA library in an appropriateexpression vector system;

[0026] (3) screening a PKG-containing cDNA library constructed in abacteriophage or plasmid vector with a labeled degenerateoligonucleotide probe designed from the amino acid sequence of the PKGprotein;

[0027] (4) screening a PKG-containing cDNA library constructed in abacteriophage or plasmid vector with a partial cDNA encoding the PKGprotein. This partial cDNA is obtained by the specific PCR amplificationof PKG DNA fragments through the design of degenerate oligonucleotideprimers from the amino acid sequence known for other kinases which arerelated to the PKG protein;

[0028] (5) screening a PKG-containing cDNA library constructed in abacteriophage or plasmid vector with a partial cDNA encoding the PKGprotein. This strategy may also involve using gene-specificoligonucleotide primers for PCR amplification of PKG cDNA identified asan EST as described above; or

[0029] (6) designing 5′ and/or 3′ gene specific oligonucleotides usingSEQ ID NO: 10 or SEQ ID NO: 12 as a template so that either the fulllength cDNA may be generated by known RACE techniques, or a portion ofthe coding region may be generated by these same known RACE techniquesto generate and isolate a portion of the coding region to use as a probeto screen one of numerous types of cDNA and/or genomic libraries inorder to isolate a full length version of the nucleotide sequenceencoding PKG.

[0030] It is readily apparent to those skilled in the art that suitablecDNA libraries may be prepared from cells or cell lines which have PKGactivity. The selection of cells or cell lines for use in preparing acDNA library to isolate a PKG cDNA may be done by first measuring cellassociated PKG activity using any known assay for PKG activity.

[0031] Preparation of cDNA libraries can be performed by standardtechniques well known in the art. Well known cDNA library constructiontechniques can be found for example, in Sambrook, et al., 1989,Molecular Cloninig: A Laboratory Manual; Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. Complementary DNA libraries may also beobtained from numerous commercial sources, including but not limited toClontech Laboratories, Inc. and Stratagene.

[0032] It is also readily apparent to those skilled in the art that DNAencoding PKG may also be isolated from a suitable genomic DNA library.Construction of genomic DNA libraries can be performed by standardtechniques well known in the art. Well known genomic DNA libraryconstruction techniques can be found in Sambrook, et al., supra. GenomicDNA libraries may also be obtained from numerous commercial sources,including but not limited to Clontech Laboratories, Inc. and Stratagene.

[0033] In order to clone the PKG gene by one of the preferred methods,the amino acid sequence or DNA sequence of PKG or a homologous proteinmay be necessary. To accomplish this, the PKG or a homologous proteinmay be purified and partial amino acid sequence determined by automatedsequenators. It is not necessary to determine the entire amino acidsequence, but the linear sequence of two regions of 6 to 8 amino acidscan be determined for the PCR amplification of a partial PKG DNAfragment. Once suitable amino acid sequences have been identified, theDNA sequences capable of encoding them are synthesized. Because thegenetic code is degenerate, more than one codon may be used to encode aparticular amino acid, and therefore, the amino acid sequence can beencoded by any of a set of similar DNA oligonucleotides. Only one memberof the set will be identical to the PKG sequence but others in the setwill be capable of hybridizing to PKG DNA even in the presence of DNAoligonucleotides with mismatches. The. mismatched DNA oligonucleotidesmay still sufficiently hybridize to the PKG DNA to permit identificationand isolation of PKG encoding DNA.

[0034] Alternatively, the nucleotide sequence of a region of anexpressed sequence may be identified by searching one or more availabledatabases. Gene-specific primers may be used to perform PCRamplification of a cDNA of interest from either a cDNA library or apopulation of cDNAs. As noted above, the appropriate nucleotide sequencefor use in a PCR-based method may be obtained from SEQ ID NO: 10 or SEQID NO: 12, either for the purpose of isolating overlapping 5′ and 3′RACE products for generation of a full-length sequence coding for PKG,or to isolate a portion of the nucleotide sequence coding for PKG foruse as a probe to screen one or more cDNA- or genomic-based libraries toisolate a full-length sequence encoding PKG or PKG-like proteins.

[0035] DNA sequences of the invention can be obtained by severalmethods. For example, the DNA can be isolated using hybridizationtechniques which are well known in the art. These include, but are notlimited to: 1) hybridization to genomic or cDNA libraries with probes todetect homologous nucleotide sequences; 2) antibody screening ofexpression libraries to detect cloned DNA fragments with sharedstructural features; and 3) PCR amplification of a desired nucleotidesequence using oligonucleotide primers. Preferably the PKGpolynucleotide of the invention is derived from a protozoal organism,and most preferably from an Eimeria species. Screening procedures whichrely on nucleic acid hybridization make it possible to isolate any genesequence from any organism, provided the appropriate probe is available.Oligonucleotide probes, which correspond to a part of the sequenceencoding the protein in question, can be synthesized chemically. Thisrequires that short, oligopeptide stretches of amino acid sequence mustbe known.

[0036] The DNA sequence encoding the protein can be deduced from thegenetic code; however, the degeneracy of the code must be taken intoaccount. It is possible to perform a mixed addition reaction when thesequence is degenerate. This includes a heterogeneous mixture ofdenatured double-stranded DNA. For such screening, hybridization ispreferably performed on either single-stranded DNA or denatureddouble-stranded DNA. Hybridization is particularly useful in thedetection of cDNA clones derived from sources where an extremely lowamount of mRNA sequences relating to the polypeptide of interest arepresent. In other words, by using stringent hybridization conditionsdirected to avoid non-specific binding, it is possible, for example, toallow the autoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Wallace, et al., Nucl. Acid Res.,9:879, 1981).

[0037] The development of specific DNA sequences encoding PKG can alsobe obtained by: 1) isolation of double-stranded DNA sequences fromgenomic DNA; 2) chemical manufacture of a DNA sequence to provide thenecessary codons for the polypeptide of interest; and 3) in vitrosynthesis of a double-stranded DNA sequence by reverse transcription ofmRNA isolated from a eukaryotic donor cell. In the latter case, adouble-stranded DNA complement of mRNA is eventually formed which isgenerally referred to as cDNA.

[0038] When the entire sequence of amino acid residues of the desiredpolypeptide is not known, the direct synthesis of DNA sequences is notpossible and the method of choice is the synthesis of cDNA sequences.Among the standard procedures for isolating cDNA sequences of interestis the formation of plasmid- or phage-carrying cDNA libraries which arederived from reverse transcription of mRNA which is abundant in donorcells that have a high level of genetic expression. When used incombination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have been denatured into asingle-stranded form.

[0039] A preferred method for obtaining genomic DNA for example is thePolymerase Chain Reaction (PCR), which relies on an in vitro method ofnucleic acid synthesis by which a particular segment of DNA isspecifically replicated. Two oligonucleotide primers that flank the DNAfragment to be amplified are utilized in repeated cycles of heatdenaturation of the DNA, annealing of the primers to their complementarysequences, and extension of the annealed primers with DNA polymerase.These primers hybridize to opposite strands of the target sequence andare oriented so that DNA synthesis by the polymerase proceeds across theregion between the primers. Since the extension products themselves arealso complementary to and capable of binding primers, successive cyclesof amplification essentially double the amount of the target DNAsynthesized in the previous cycle. The result is an exponentialaccumulation of the specific target fragment, approximately 2<n>, wheren is the number of cycles of amplification performed (see PCR Protocols,Eds. Innis, et al., Academic Press, Inc., 1990, incorporated herein byreference).

[0040] A cDNA expression library, in a vector such as lambda gt11, canbe screened indirectly for PKG peptides having at least one epitope,using antibodies specific for PKG. Such antibodies can be eitherpolyclonally or monoclonally derived and used to detect an expressionproduct indicative of the presence of a PKG cDNA. The polynucleotidesequence for PKG also includes sequences complementary to thepolynucleotide encoding PKG (antisense sequences). Antisense nucleicacids are DNA or RNA molecules that are complementary to at least aportion of a specific mRNA molecule (Weintraub, Scientific American,262:40, 1990). The invention embraces all antisense polynucleotidescapable of inhibiting the production of the PKG polypeptide. In thecell, the antisense nucleic acids hybridize to the corresponding mRNA,forming a double-stranded molecule. The antisense nucleic acids mayinterfere with the translation of the mRNA since the cell will nottranslate a mRNA that is double-stranded, or alternatively, thedouble-stranded mRNA is targeted for degradation. Antisense oligomers ofabout 15 nucleotides are preferred, since they are easily synthesized,small enough to enter the cell, and are less likely to cause problemsthan larger molecules when introduced into the target PKG-producingcell. The use of antisense methods to inhibit the translation of genesis well known in the art (Marcus-Sakura, Anal. Biochem., 172:289, 1988).

[0041] Vectors, Host Cells, Expression

[0042] DNA sequences encoding PKG can be expressed in vitro by DNAtransfer into a suitable host cell. “Host cells” are cells in which avector carrying DNA sequences of interest can be propagated and theprotein coded for by the DNA sequences can be expressed. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used. Methods of stable transfer,meaning that the foreign DNA is continuously maintained in the host, areknown in the art.

[0043] A variety of expression vectors may be used to expressrecombinant PKG in host cells. “Expression vectors” are DNA sequencesthat are required for the transcription of cloned DNA and thetranslation of their mRNAs in an appropriate host. Such vectors can beused to express eukaryotic DNA in a variety of hosts such as bacteria,blue green algae, plant cells, insect cells, apicomplexan parasites andanimal cells. Specifically designed vectors allow the shuttling of DNAbetween hosts such as bacteria-yeast, bacteria-insect cells,bacteria-apicomplexan parasites or bacteria-animal cells. Anappropriately constructed expression vector should contain: an origin ofreplication for autonomous replication in host cells, selectablemarkers, a limited number of useful restriction enzyme sites, apotential for high copy number, and active promoters. Other expressionvectors do not contain an origin of replication for autonomousreplication in host cells but rather depend on the ability of the vectorto stably integrate (either randomly or by a homologous integrationevent) using a marker to select for integration/maintenance. A promoteris defined as a DNA sequence that directs RNA polymerase to bind to DNAand initiate RNA synthesis. A strong promoter is one which causes mRNAsto be initiated at high frequency. Expression vectors may include, butare not limited to, cloning vectors, modified cloning vectors,specifically designed plasmids or viruses.

[0044] Commercially available mammalian expression vectors which may besuitable for recombinant PKG expression, include but are not limited to,pcDNA3, pcDNA3.1, pcDNAI, pcDNAlamp (Invitrogen), pMC1neo (Stratagene),pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593)pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt(ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC37460), and λZD35 (ATCC 37565).

[0045] A variety of bacterial expression vectors may be used to expressrecombinant PKG in bacterial cells. Commercially available bacterialexpression vectors which may be suitable for recombinant PKG expressioninclude, but are not limited to pCR2.1 (Invitrogen), pET11a (Novagen),lambda gt11 (Invitrogen), pcDNAII (Invitrogen), pKK223-3 (Pharmacia),and pQE vectors (Qiagen).

[0046] A variety of fungal cell expression vectors may be used toexpress recombinant PKG in fungal cells. Commercially available fungalcell expression vectors which may be suitable for recombinant PKGexpression include but are not limited to pYES2 (Invitrogen), Pichiaexpression vector (Invitrogen).

[0047] A variety of insect cell expression vectors may be used toexpress recombinant PKG in insect cells. Commercially available insectcell expression vectors which may be suitable for recombinant expressionof PKG include but are not limited to pBlueBacIII, pBlueBacHis2(Invitrogen) and pFastBac1, pFastBacHT (Life Technologies).

[0048] A variety of expression vectors may be used to expressrecombinant PKG in apicomplexan parasites, most notably Toxoplasmagondii. Expression vectors which may be suitable for recombinantexpression of PKG include but are not limited to pminCAT/HXGPRT-,pDHFR-TSc3/M3, pDHFR-TSc3/M2M3, pminiHXGPRT, and pminP30/G (NIH AIDSResearch and Reference Reagent Program).

[0049] The expression vector may be introduced into host cells via anyone of a number of techniques including but not limited to infection,transformation, transfection, lipofection, protoplast fusion, andelectroporation. The expression vector-containing cells are clonallypropagated and individually analyzed to determine whether they producePKG protein. Identification of PKG expressing host cell clones may bedone by several means, including but not limited to immunologicalreactivity with anti-PKG antibodies.

[0050] Expression of PKG DNA may also be performed using in vitroproduced synthetic mRNA or native mRNA. Synthetic mRNA or mRNA isolatedfrom PKG producing cells can be efficiently translated in variouscell-free systems, including but not limited to wheat germ extracts andreticulocyte extracts, as well as efficiently translated in cell basedsystems, including but not limited to microinjection into frog oocytes,with microinjection into frog oocytes being preferred.

[0051] A variety of host-expression vector systems may be utilized toexpress the PKG coding sequence. These include but are not limited tomicroorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining the PKG coding sequence; yeast transformed with recombinantyeast expression vectors containing the PKG coding sequence; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing the PKG coding sequence; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing the PKG coding sequence; or animal cell systems infected withrecombinant virus expression vectors (e.g., retroviruses, adenovirus,vaccinia virus) containing the PKG coding sequence, or transformedanimal cell systems engineered for stable expression.

[0052] To determine the PKG cDNA sequence(s) that yields optimal levelsof PKG protein, PKG cDNA molecules including but not limited to thefollowing can be constructed: the full-length open reading frame of thePKG cDNA and various constructs containing portions of the cDNA encodingonly specific domains of the protein or rearranged domains of theprotein. All constructs can be designed to contain none, all or portionsof the 5′ and/or 3′ untranslated region of PKG. PKG activity and levelsof protein expression can be determined following the introduction, bothsingly and in combination, of these constructs into appropriate hostcells. Following determination of the PKG cDNA cassette yielding optimalexpression in transient assays, this PKG cDNA construct is transferredto a variety of expression vectors (including recombinant viruses),including but not limited to those for mammalian cells, plant cells,insect cells, oocytes, bacteria, and yeast cells.

[0053] The protein coding region of the recombinant PKG coding sequencemay also be modified at the level of the cDNA to add epitope tags.Modification of the cDNA sequence is usually but not exclusivelydirected to the amino- or carboxy-terminal end of the protein codingregion. This is accomplished by any of several methods common in theart. Examples of modifications include but are not limited to theinfluenza viral HA tag, the FLAG tag, a hexahistidine tag, the c-myctag, as well as fusions with maltose binding protein, glutathionetransferase and green fluorescent protein. These tags can serve as ameans to distinguish recombinant PKG from host PKG immunologically usingcommercially available antisera to the tag sequences and as apurification tool to resolve recombinant from host PKGs.

[0054] Levels of recombinant PKG protein in host cells is quantified bya variety of techniques including, but not limited to, immunoaffinityand/or ligand affinity techniques. PKG-specific affinity beads orPKG-specific antibodies are used to isolate PKG protein metabolicallylabeled with radioactive amino acids such as [³H]-leucine or[³⁵S]-methionine or unlabelled PKG protein. Labeled PKG protein isanalyzed by SDS-PAGE. Unlabelled PKG protein is detected by Westernblotting, ELISA or RIA assays employing PKG-specific or epitopetag-speecific antibodies.

[0055] Purification of PKG

[0056] Following expression of PKG in a host cell, PKG protein may berecovered to provide PKG in active form. Several PKG purificationprocedures are available and suitable for use. Recombinant PKG may bepurified from cell lysates and extracts, or from conditioned culturemedium, by various combinations of, or individual application of saltfractionation, ion exchange chromatography, size exclusionchromatography, hydroxyapatite adsorption chromatography, hydrophobicinteraction chromatography and cyclic nucleotide affinitychromatography.

[0057] In addition, recombinant PKG can be separated from other cellularproteins by use of an immuno-affinity column made with monoclonal orpolyclonal antibodies specific for full length PKG, or polypeptidefragments of PKG. Additionally, polyclonal or monoclonal antibodies maybe raised against a synthetic peptide (usually from about 9 to about 25amino acids in length) from a portion of the protein.

[0058] Monospecific antibodies to PKG are purified from mammalianantisera containing antibodies reactive against PKG or are prepared asmonoclonal antibodies reactive with PKG using the technique of Kohlerand Milstein (1975, Nature 256: 495-497). Monospecific antibody as usedherein is defined as a single antibody species or multiple antibodyspecies with horogenous binding characteristics for PKG. Homogenousbinding as used herein refers to the ability of the antibody species tobind to a specific antigen or epitope, such as those associated with thePKG, as described above. PKG specific antibodies are raised byimmunizing animals such as mice, rats, guinea pigs, rabbits, goats,horses and the like, with an appropriate concentration of PKG or PKGsynthetic peptide either with or without an immune adjuvant.

[0059] Preimmune serum is collected prior to the first immunization.Each animal receives between about 0.1 μg and about 1000 μg of PKGassociated with an acceptable immune adjuvant. Such acceptable adjuvantsinclude, but are not limited to, Freund's complete, Freund's incomplete,alum-precipitate, water in oil emulsion containing Corynebacteriumparvum and tRNA. The initial immunization consists of the PKG protein orPKG synthetic peptide in, preferably, Freund's complete adjuvant atmultiple sites either subcutaneously (SC), intraperitoneally (IP) orboth. Each animal is bled at regular intervals, preferably weekly, todetermine antibody titer. The animals may or may not receive boosterinjections following the initial immunization. Those animals receivingbooster injections are generally given an equal or smaller amount of PKGin Freund's incomplete adjuvant by the same route. Booster injectionsare given at about three week intervals until maximal titers areobtained. At about 7 days after each booster immunization or aboutweekly after a single immunization, the animals are bled, the serumcollected, and aliquots are stored at about −20° C.

[0060] Monoclonal antibodies (mAb) reactive with PKG are prepared byimmunizing inbred mice, preferably Balb/c, with PKG. The mice areimmunized by the IP or SC route with about 1 μg to about 100 μg,preferably about 10 μg, of PKG in about 0.5 ml buffer or salineincorporated in an equal volume of an acceptable adjuvant, as discussedabove. Freund's complete adjuvant is preferred. The mice receive aninitial immunization on day 0 and are rested for about 3 to about 30weeks. Immunized mice are given one or more booster immunizations ofabout 1 to about 100 μg of the same PKG antigen in a buffer solutionsuch as phosphate buffered saline by the intravenous (IV) route.Lymphocytes, from antibody positive mice, preferably spleniclymphocytes, are obtained by removing spleens from immunized mice bystandard procedures known in the art. Hybridoma cells are produced bymixing the splenic lymphocytes with an appropriate fusion partner,preferably myeloma cells, under conditions which will allow theformation of stable hybridomas. Fusion partners may include, but are notlimited to: mouse myelomas P3/NS1/Ag 4-1; MPC-11; S-194 and Sp 2/0, withSp 2/0 being preferred. The antibody producing cells and myeloma cellsare fused in polyethylene glycol, about 1000 mol. wt., at concentrationsfrom about 30% to about 50%. Fused hybridoma cells are selected bygrowth in hypoxanthine, thymidine and aminopterin supplementedDulbecco's Modified Eagles Medium (DMEM) by procedures known in the art.Supernatant fluids are collected from growth positive wells on aboutdays 14, 18, and 21 and are screened for antibody production by animmunoassay such as solid phase immunoradioassay (SPIRA) using PKG asthe antigen. The culture fluids are also tested in the Ouchterlonyprecipitation assay to determine the isotype of the mAb. Hybridoma cellsfrom antibody positive wells are cloned by a technique such as the softagar technique of MacPherson, 1973, Soft Agar Techniques, in TissueCulture Methods and Applications, Kruse and Paterson, Eds., AcademicPress.

[0061] Monoclonal antibodies are produced in vivo by injection ofpristine primed Balb/c mice, approximately 0.5 ml per mouse, with about2×10⁶ to about 6×10⁶ hybridoma cells about 4 days after priming. Ascitesfluid is collected at approximately 8-12 days after cell transfer andthe monoclonal antibodies are purified by techniques known in the art.

[0062] In vitro production of anti-PKG monoclonal antibody is carriedout by growing the hydridoma in DMEM containing about 2% fetal calfserum to obtain sufficient quantities of the specific monoclonalantibody. The monoclonal antibodies are purified by techniques known inthe art.

[0063] Antibody titers of ascites or hybridoma culture fluids aredetermined by various serological or immunological assays which include,but are not limited to, precipitation, passive agglutination,enzyme-linked immunosorbent antibody (ELISA) technique andradioimmunoassay (RIA) techniques. Similar assays are used to detect thepresence of PKG in body fluids or tissue and cell extracts.

[0064] It is readily apparent to those skilled in the art that the abovedescribed methods for producing monospecific antibodies may be utilizedto produce antibodies specific for PKG polypeptide fragments, orfull-length PKG polypeptide.

[0065] PKG antibody affinity columns are made by adding the antibodiesto Affigel-10 (Biorad), a gel support which is pre-activated withN-hydroxysuccinimide esters such that the antibodies form covalentlinkages with the agarose gel bead support. The antibodies are thencoupled to the gel via amide bonds with the spacer arm. The remainingactivated esters are then quenched with 1M ethanolamine HCl (pH 8). Thecolumn is washed with water followed by 0.23 M glycine HCl (pH 2.6) toremove any non-conjugated antibody or extraneous protein. The column isthen equilibrated in phosphate buffered saline (pH 7.3) and the cellculture supernatants or cell extracts containing PKG or PKG fragmentsare slowly passed through the column. The column is then washed withphosphate buffered saline until the optical density (A₂₈₀) falls tobackground, then the protein is eluted with 0.23 M glycine-HCl (pH 2.6).The purified PKG protein is then dialyzed against phosphate bufferedsaline.

[0066] One skilled in the art will appreciate that the above describedprocedures for purification of recombinant expressed PKG are alsosuitable for purification of the native enzyme from a protozoanparasite.

[0067] Inhibitors—Assays and Molecules

[0068] In another aspect of the present invention there is provided amethod for identifying compounds having antiprotozoal activity. In oneembodiment said method comprises:

[0069] (a) contacting protozoal PKG with (i) a known amount of a labeledcompound that interacts with a PKG and (ii) a known dilution of a testcompound or a natural product extract; and

[0070] (b) quantitating the percent inhibition of interaction of saidlabeled compound induced by said test compound.

[0071] The PKG may be a purified or partially purified native enzyme, acloned PKG or an engineered variant thereof, a crude preparation of theenzyme, or an extract containing PKG activity. Fragments of PKG thatretain the desired enzyme activity are also within the scope of theinvention.

[0072] A compound that interacts with PKG may be one that is a substratefor the enzyme, or one that binds to the enzyme either at its activesite or at an alternate site (e.g., the cGMP binding domains) thatresults in altered enzyme activity. A substrate may be a generic proteinkinase substrate (e.g., myosin basic protein, MBP), a synthetic peptideor other naturally occurring substrates. Examples of compounds that bindto PKG are known inhibitors such as KT5823, synthetic or naturallyoccurring peptides as well as other small molecule (i.e. non-peptidyl)inhibitors such as2-(4-fluorophenyl)-5-(N-methylpiperidin-4-yl)-3-(4-pyridyl)pyrrole,which will be referred to herein as “Inhibitor Compound”. The InhibitorCompound and its preparation are disclosed in U.S. Pat. No. 5,792,778.The compound that interacts with PKG is preferably labeled to allow easyquantitation of the level of interaction between the compound and theenzyme. Preferred radiolabels are [¹⁴C] or [³H]. The tritiated InhibitorCompound may be prepared as follows:

[0073] A solution of Inhibitor Compound (as the free amine, 2.5 mg,0.007 mmol) in DMSO (0.8 ml) was stirred for half-hour at roomtemperature. High specific activity tritium labeled methyl iodide (200mCi, 0.0026 mmol, at 80 Ci/mmol in 0.4 ml toluene solution) was added tothis mixture and the resulting clear solution was stirred for 24 hour.The product was diluted with isopropanol (35 mL) evaporated to neardryness and dissolved with 3 mL of methanol for HPLC purification. Theproduct was collected into a vial containing 7 mL of cold water (chilledin the refrigerator before use). After assay for radioactivity andradio-purity, the sample was stored in the refrigerator to preventdecomposition. Total activity of the purified product was 45 mCi and atspecific activity of 68 Ci/mmol (by UV spectrum measurement). HPLCconditions are: Zorbax SB-C18 Semi-prep column (5 um, 9.4 mmI.D.×250mm), 65/35/0.1 (v/v/v) water/acetonitrile/HClO4. UV-detection at 230 nm,4.5 mL/min, Rt=24.5 min, radiochemical purity: 98.5%

[0074] The test compound may be a synthetic compound, a purifiedpreparation, crude preparation, or an initial extract of a naturalproduct obtained from plant, microorganism or animal sources.

[0075] One particular embodiment of the present method is based on testcompound induced inhibition of PKG activity. The enzyme inhibition assayinvolves adding PKG or an extract containing PKG to mixtures of enzymesubstrates (one of which is radiolableled) and the test compound, eachof which are present in known concentrations. The amount of the enzymeis chosen such that less than 20% of the radiolabeled substrate (usually[³²P]- or [³³P]-ATP,) is consumed during the assay. The assay is carriedout with the test compound at a series of different dilution levels.After a period of incubation, the labeled portion of the ATP substratebecomes covalently attached by enzymatic action to the peptide orprotein substrate. This reaction product is separated fromunincorporated precursor and counted. The assay is generally carried outin parallel with a control (no test compound) and a positive control(containing a known enzyme inhibitor instead of a test compound). Theconcentration of the test compound at which 50% of the enzyme activityis inhibited (IC₅₀) is determined using a recognized method.

[0076] Although enzyme inhibition is the most direct measure of theinhibitory activity of the test compound, the present inventors havefound that results obtained from competitive binding assay in which thetest compound competes with a known inhibitor for binding to the enzymeactive site correlate well with the results obtained from enzymeinhibition assay described above. Accordingly, another particularembodiment of the present method is based on competitive binding of testcompound and a known PKG inhibitor. The binding assay represents a moreconvenient way to assess enzyme inhibition since it allows the use of acrude extract containing PKG rather than partially purified enzyme. Theuse of a crude extract may not always be suitable in the enzymeinhibition assay because other enzymes present in the extract mayphosphorylate the test substrate. The competition binding assay iscarried out by adding the PKG or an extract containing PKG activity to amixture of the test compound and a labeled inhibitor, both of which arepresent in the mixture in known concentrations. After incubation, theenzyme-inhibitor complex is separated from the unbound labeledinhibitors and unlabeled test compound, and counted. The concentrationof the test compound required to inhibit 50% of the binding of thelabeled inhibitor to the PKG (IC₅₀) is calculated.

[0077] In a preferred embodiment, the method of the present inventionutilizes a recombinant PKG, a native PKG, or an extract containing PKGobtained from a protozoal source, such as Eimeria, Toxoplasma orPlasmodiurn sp.

[0078] In a more preferred embodiment, the method of the presentinvention further comprises determining the IC₅₀ of test compoundsagainst host PKG in either the enzyme inhibition assay or the bindingassay as described above, to identify those compounds that haveselectivity for parasitic PKG over that of a host. The assays are thesame as previously described, with the PKG activity obtained from a hostof protozoa; for example the host PKG may be obtained from a mammaliansource, e.g. human, or an avian source, e.g. chicken.

[0079] Another method useful to identify inhibitors that are selectivefor parasitic PKG is the use of an in gel kinase assay to determine thelevel of substrate phosphorylation catalyzed by parasite PKG relative tohost PKG or other cellular kinase activities. Thus compounds thatspecifically inhibit phosphorylation of a test substrate (e.g., myelinbasic protein) by parasite PKG and not a host PKG, would be consideredselective parasitic PKG inhibitors.

[0080] Where the enzyme inhibition or binding assay utilizes a crudepreparation or an extract containing PKG, the target of the testcompound may be verified by examining the level of native substratephosphorylation. Thus, the intact host or parasitic cell containing theenzyme is treated with the test compound. Alternatively, intact host orparasite cells containing the enzyme are treated with test compound inthe presence of labeled phosphate ([³³P] or [³²P] is the preferredlabel). In both cases the cells are lysed, soluble proteins arepartially purified, and analyzed by two dimensional polyacrylamideelectrophoresis. Proteins are detected by staining or by detection ofradiolabel by autoradiography or fluorography. Differentiallyphosphorylated species can readily be distinguished on such gels due tothe altered migration of multiply phosphorylated proteins or by thepresence/absence of an autoradiographic signal. A PKG inhibitor willblock the incorporation of radiolabeled phosphate into substrate. Sincethis technique uses intact cells treated with the test compound, thistechnique may also be used to identify prodrugs that may be converted toa PKG inhibitor within the cellular environment, but may not be soidentified by assay based on the enzyme itself.

[0081] Another embodiment of the method for identifying compounds havingantiprotozoal activity comprises:

[0082] (a) contacting an intact host or protozoal cell with a testcompound or a natural product extract;

[0083] (b) disrupting said cell to obtain a biochemical fractionpossessing PKG catalytic activity; and

[0084] (c) determining the level of PKG activity in said biochemicalfraction.

[0085] Thus intact host cell(s) are treated with a test compound at aknown concentration (or a natural product extract at know dilution) for1 minute to 12 hours. Thereafter the host cells are lysed, for example,using the method described in Example 3 or other known methods in theart. The level of PKG catalytic activity may be determined using methodshereinafter described in the Examples as well as other methods generallyknown by those skilled in the art.

[0086] Compounds identified as PKG inhibitors may be useful asantiprotozoal agents, and as such, they may be used in the treatment andprevention of protozoal diseases in human and animals, includingpoultry. Thus, PKG inhibitors may be administered to a host sufferingfrom a protozoal infection a therapeutically effective amount of acompound which inhibits PKG. A therapeutically effective amount may beone that is sufficient to inhibit PKG of the causative protozoa.Examples of protozoal diseases against which PKG inhibitors may be used,and their respective causative pathogens, include: 1) amoebiasis(Dientamoeba sp., Entanioeba histolytica); 2) giardiasis (Giardialamblia); 3) malaria (Plasmodium species including P. vivax, P.falciparuni, P. malariae and P. ovale); 4) leishmaniasis (Leishliianiaspecies including L. donovani, L. tropica, L. mexicana, and L.braziliensis); 5) trypanosomiasis and Chagas disease (Trypanosomaspecies including T. brucei, T. theileri, T. rhodesiense, T. gambiense,T. evansi, T. equiperdum, T. equinuni, T. congolense, T. vivax and Tcruzi); 6) toxoplasmosis (Toxoplasma gondii); 7) neosporosis (Neosporacaniiuni); 8) babesiosis (Babesia sp.); 9) cryptosporidiosis(Cryptosporidium sp.); 10) dysentary (Balantidium coli); 11) vaginitis(Trichomonas species including T.vaginitis, and T. foetus); 12)coccidiosis (Fimeria species including E. tenella, E. necatrix, E.acervulina, E. maxima, E. brunetti, E. mitis, E. bovis, E. melagramatis,and Isospora sp.); 13) enterohepatitis (Histomonas gallinarum), and 14)infections caused by Anaplasma sp., Besnoitia sp., Leucocytozoan sp.,Microsporidia sp., Sarcocystis sp., Theileria sp., and Pneumocystiscarinii.

[0087] PKG inhibitors are preferably used in the treatment or preventionof protozoal infections caused by a member of the sub-phyllumApicomplexa. PKG inhibitors are also preferably used in the treatment orprevention of malaria, toxoplasmosis, cryptosporidiosis andtrypanosomiasis in humans and animals; and in the management ofcoccidiosis, particularly in poultry, either to treat coccidialinfection or to prevent the occurrence of such infection.

[0088] In the case that a PKG inhibitor is expected to be administeredon a chronic basis, such as in the prevention of coccidiosis in poultry,the PKG inhibitor preferably is selective for protozoal over the hostPKG activity. Long term administration of such a selective inhibitorwould minimize adverse effects to the host due to PKG inhibition.

[0089] Two specific examples of using PKG inhibitors to prevent theestablishment of parasitic infections in humans and animals are 1) theprevention of Plasniodiunt (malaria) infection in humans in endemicareas and 2) the prevention of coccidiosis in poultry by administeringthe compound continuously in the feed or drinking water. Malaria is thenumber one cause of death in the world. The disease is transmitted bymosquitoes in endemic areas and can very rapidly progress to a lifethreatening infection. Therefore, individuals living in or visitingareas where malaria carrying mosquitoes are present routinely takeprophylactic drugs to prevent infection. The PKG inhibitor would beadministered orally or parenterally one or more time(s) a day. The dosewould range from 0.01 mg/kg to 100 mg/kg. The compound could beadministered for the entire period during which the patient or animal isat risk of acquiring a parasitic infection.

[0090] Coccidiosis is a disease which can occur in humans and animalsand is caused by several genera of coccidia. The most economicallyimportant occurrence of coccidiosis is the disease in poultry.Coccidiosis in poultry is caused by protozoal parasites of the genusEimeria. The disease can spread quite rapidly throughout flocks of birdsvia contaminated feces. The parasites destroy gut tissue and thereforedamage the gut lining impairing nutrient absorption. An outbreak ofcoccidiosis in a poultry house can cause such dramatic economic lossesfor poultry producers that it has become standard practice to useanticoccidial agents prophylactically in the feed. A PKG inhibitor wouldbe administered in the feed or drinking water for a portion of, or theentire life of the birds. The dose would range between 0.1 ppm to 500ppm in the feed or water.

[0091] For treatment of established parasitic infections in humans oranimals, the PKG inhibitor could be administered orally or parenterallyonce the infection is suspected or diagnosed. The treatment period wouldvary according to the specific parasitic disease and the severity of theinfection. In general the treatment would be continued until theparasites were eradicated and/or the symptoms of the disease wereresolved. Two specific examples are the treatment of a 1)Cryptosporidium parvum infection in an animal or human and treatment ofacute Plasmodium falciparum malaria in humans. Cryptosporidium parvum isa protozoal parasite that infects and destroys cells lining theintestinal tract of humans and animals. The infection establishes quiterapidly and has acute effects on the patient. In the case of humans,patients get severe dysentery for a period of 5-7 days. In immunecompromised patients C. parvum infections can persist and can be lifethreatening. In animals C. parvum infection is the number one cause ofdeath in young dairy calves. A C. parvum infection can be easilydiagnosed by symptoms and examination of a stool sample. Once thedisease is suspected and/or diagnosed, treatment with a PKG inhibitorcan be initiated. The dose would vary between 0.01 mg/kg to 500 mg/kg.Treatments would be one or more time(s) a day, orally or parenterally,until the infection is eliminated. Routinely this dosing period would be1-3 weeks.

[0092]P. falciparum causes acute life threatening malarial infections inhumans. The infection if left untreated can quite often result in deathof the patient. A malaria infection can be easily diagnosed by symptomsand examination of a blood sample from the patient. Treatment would beinitiated following diagnosis. A PKG inhibitor would be administered oneor more time(s) a day, orally or parenterally, until the infection waseliminated. The dose would range between 0.01 mg/kg to 200 mg/kg.

[0093] PKG inhibitors may be administered to a host in need of treatmentin a manner similar to that used for other antiprotozoal agents; forexample, they may be administered parenterally, orally, topically, orrectally. The dosage to be administered will vary according to theparticular compound used, the infectious organism involved, theparticular host, the severity of the disease, physical condition of thehost, and the selected route of administration; the appropriate dosagecan be readily determined by a person skilled in the art. For thetreatment of protozoal diseases in human and animals, the dosage mayrange from 0.01 mg/kg to 500 mg/kg. For prophylactic use in human andanimals, the dosage may range from 0.01 mg/kg to 100 mg/kg. For use asan anticoccidial agent, particularly in poultry, the compound ispreferably administered in the animals' feed or drinking water. Thedosage ranges from 0.1 ppm to 500 ppm.

[0094] PKG inhibitors may be formulated according to conventionalpharmaceutical compounding techniques. Thus a PKG inhibitor compositionmay contain, in addition to the active ingredient, a pharmaceuticallyacceptable carrier and optionally other therapeutic ingredients. Thecomposition may be one that is suitable for oral, rectal, topical, orparenteral (including subcutaneous, intramuscular, and intravenous)administrations, although the most suitable route in any given case willdepend on the particular host, and nature and severity of the conditionsfor which the active ingredient is being administered. The compositionmay be conveniently presented in unit dosage form and prepared by any ofthe methods well-known in the art of pharmacy.

[0095] Pharmaceutical compositions of the present invention suitable fororal administration may be presented as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient, as a powder or granules or as a solution or a suspension inan aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or awater-in-oil liquid emulsion. Such compositions may be prepared by anyof the methods of pharmacy but all methods include the step of bringinginto association the active ingredient with the carrier whichconstitutes one or more necessary ingredients. In general, thecompositions are prepared by uniformly and intimately admixing theactive ingredient with liquid carriers or finely divided solid carriersor both, and then, if necessary, shaping the product into the desiredpresentation. For example, a tablet may be prepared by compression ormolding, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing, in a suitable machine, theactive ingredient in a free-flowing form such as powder or granules,optionally mixed with a binder, lubricant, inert diluent, surface activeor dispersing agent. Molded tablets may be made by molding in a suitablemachine, a mixture of the powdered compound moistened with an inertliquid diluent. Desirably, each tablet contains from about 1 mg to about500 mg of the active ingredient and each cachet or capsule contains fromabout 1 to about 500 mg of the active ingredient.

[0096] Pharmaceutical compositions of the present invention suitable forparenteral administration may be prepared as solutions or suspensions ofthese active compounds in water suitably mixed with a surfactant such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

[0097] The pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g. glycerol, propylene glycol and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

[0098] Suitable topical formulations include transdermal devices,aerosols, creams, ointments, lotions, dusting powders, and the like.These formulations may be prepared via conventional methods containingthe active ingredient. To illustrate, a cream or ointment is prepared bymixing sufficient quantities of hydrophilic material and water,containing from about 5-10% by weight of the compound, in sufficientquantities to produce a cream or ointment having the desiredconsistency.

[0099] Pharmaceutical compositions suitable for rectal administrationwherein the carrier is a solid are most preferably presented as unitdose suppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art, and the suppositories may beconveniently formed by admixture of the combination with the softened ormelted carrier(s) followed by chilling and shaping moulds.

[0100] It should be understood that in addition to the aforementionedcarrier ingredients the pharmaceutical formulations described above mayinclude, as appropriate, one or more additional carrier ingredients suchas diluents, buffers, flavoring agents, binders, surface-active agents,thickeners, lubricants, preservatives (including anti-oxidants) and thelike, and substances included for the purpose of rendering theformulation isotonic with the blood of the intended recipient.

[0101] For use in the management of coccidiosis in poultry, a PKGinhibitor may be conveniently administered as a component of a feedcomposition. Suitable poultry feed composition will typically containfrom about 1 ppm to about 1000 ppm, preferably from about 0.01% to about0.1% percent, by weight of a PKG inhibitor. The optimum levels willnaturally vary with the species of Eimeria involved, and can be readilydetermined by one skilled in the art. Levels of in poultry feed of fromabout 0.01% to about 0.1% percent by weight of the diet are especiallyuseful in controlling the pathology associated with E. tenella, whilethe preferred concentration for similar control of intestinal-dwellingspecies is from about 0.01% to about 0.1% percent by weight of the diet.Amounts of about 0.01% to about 0.1% percent by weight are advantageousin reducing the pathogenic effects of both cecal and intestinalcoccidiosis.

[0102] In the preparation of poultry feed, a PKG inhibitor may bereadily dispersed by mechanically mixing the same in finely ground formwith the poultry feedstuff, or with an intermediate formulation (premix)that is subsequently blended with other components to prepare the finalpoultry feedstuff that is fed to the poultry. Typical components ofpoultry feedstuffs include molasses, fermentation residues, corn meal,ground and rolled oats, wheat shorts and middlings, alfalfa, clover andmeat scraps, together with mineral supplements sucheas bone meal,calcium carbonate and vitamins.

[0103] Compositions containing a compound may also be prepared in powderor liquid concentrate form. In accordance with standard veterinaryformulation practice, conventional water soluble excipients, such aslactose or sucrose, may be incorporated in the powders to improve theirphysical properties. Thus particularly suitable powders of thisinvention comprise 50 to 100% w/w, and preferably 60 to 80% w/w of thecombination and 0 to 50% w/w and preferably 20 to 40% w/w ofconventional veterinary excipients. These powders may either be added toanimal feedstuffs, for example by way of an intermediate premix, ordiluted in animal drinking water.

[0104] Liquid concentrates of this invention suitably contain awater-soluble compound combination and may optionally include aveterinarily acceptable water miscible solvent, for example polyethyleneglycol, propylene glycol, glycerol, glycerol formal or such a solventmixed with up to 30% v/v of ethanol. The liquid concentrates may beadministered to the drinking water of animals, particularly poultry.

[0105] The following examples are provided to illustrate the inventionare not to be construed as limiting the scope of the invention in anymanner.

EXAMPLE 1

[0106] Competitive Binding Assay Using [³H] Inhibitor Compound

[0107] PKG is incubated with appropriately diluted [³H] InhibitorCompound for 60 minutes on ice and free ligand is separated from boundligand by elution through 0.8 ml G-25 gel filtration columns (purchasedfrom AGTC). The void volume, containing ligand-bound protein, iscollected and radioactivity assessed by liquid scintillation counting.For competitive binding studies, excess or appropriately diluted coldcompetitor(s) are added simultaneously with, or 0.01-5 minutes before,the addition of radiolabeled compound. The concentration of the testcompound required to inhibit 50% (IC₅₀) of the binding of the labeledinhibitor to the parasite drug binding protein(s) is calculated.

[0108] For the competitive binding assay PKG may be the S100 fraction,crude preparation containing PKG activity, or recombinant PKG.Generally, the PKG preparation contains 20-50 μg of protein.

EXAMPLE 2

[0109] PKG Catalytic Activity.

[0110]E. tenella PKG purified fraction (obtained as described in Example3) (0.05 μl) is incubated for 2 hours at room temperature in 20 μl of anincubation mixture containing: 25 mM HEPES, pH 7.4, 10 mM MgCl₂, 5 mMbeta-mercaptoethanol, 20 mM beta-glycerophosphate (or 100 μM sodiumorthovanadate), 10 μM cGMP, 2 μM ATP, 10 nM [³³P] ATP (2000 Ci/mmole),400 μM Kemptide (or 2 mM Kemptide or 0.43 mg/ml myelin basic protein).The reaction is then quenched with 2.5 μl of 0.25 M phosphoric acid per20 μl assay volume, and the mixture is spotted on Whatman P81chromatography paper. The chromatography paper is allowed to dry for 2minutes, washed 5 times for 3 min. each with 150 mls of 75 mM phosphoricacid, then washed briefly with 95% ethanol. After air drying, labeledkemptide remaining on the filter is quantitated by liquid scintillationcounting.

EXAMPLE 3

[0111] Purification of the [³H] Inhibitor Compound Binding Protein fromE. tenella

[0112] (a) Preparation of the E. tenella Lysates.

[0113] Approximately 6×10⁹ E. tenella unsporulated oocysts were washedtwice in 50 mls of 10 mM Hepes pH 7.4. Oocysts were then suspended in anequal volume of lysis buffer (20% glycerol, 10 mM Hepes, pH 7.4, 0.1 mMsodium orthovanadate, protease inhibitor cocktail, [Sigma P8340, dilutedat 1:200]) and an equal volume of 3 mm glass beads and mixed byvortexing at 4° C. for 15 minutes. Oocysts were examined microscopicallyfor breakage; mixing continued until 95% of the oocysts were broken.Glass beads were then removed and washed with 20 mls of lysis bufferwhich was added to the rest of the suspension. The resulting homogenatewas separated from the glass beads and centrifuged at 100,000×g for 1hr. The pellet was discarded and the supernatant, referred to as the E.tenella S100 fraction, was retained for binding assays and furthertarget purification. After estimating the protein concentration, theS100 fraction was aliquoted and frozen at −80° C.

[0114] (b) Purification of an E. tenella [ ³H]-Inhibitor CompoundBinding Protein.

[0115] The S100 fraction was dialyzed against 2L of Buffer A (30 mMsodium phosphate, pH 7.4, 1 mM DTT, 1 mM EDTA, 20% glycerol, 10 mMsodium fluoride, 0.1 mM sodium orthovanadate) overnight at roomtemperature. The dialysate was centrifuged at 100,000×g for 1 hour andthe pellet discarded. The supernatant was applied to a HiLoad Q 26/10column (Pharmacia) that had been equilibrated in Buffer A. The columnwas washed with buffer A until the OD at 280 nm had returned to baselineindicating that no further protein was eluting from the column. Theproteins were then eluted using a linear sodium chloride gradient (0-1 MNaCl) in Buffer A. Each fraction was tested for binding using [³H]Inhibitor Compound (described in Example 1 above). The binding activityeluted as a single peak and fractions containing binding activity werepooled and dialyzed overnight against 2L Buffer B (30 mM sodiumphosphate, pH 7.4, 1 mM DTT, 1 mM EDTA, 10 mM sodium fluoride, 0.1 mMsodium orthovanadate, 1 M ammonium sulfate). The dialysed pool wasapplied to a butyl sepharose column (Pharmacia) that had beenequilibrated in Buffer B. The column was washed with buffer B until theOD at 280 nm had returned to baseline indicating that no further proteinwas eluting from the column. The proteins were eluted from the columnwith a linear inverse gradient of ammonium sulfate (from 1 M to noammonium sulfate) in Buffer B. Each fraction was tested for [³H]Inhibitor Compound binding as above and the fractions containing bindingactivity pooled and dialyzed overnight against 2L Buffer C (10 mM sodiumphosphate, pH 7.4, 1 mM DTT, 10 mM sodium fluoride, 0.1 mM sodiumorthovanadate). The dialysate was applied to a hydroxyapatite column(Biorad) that had been equilibrated in Buffer C. The column was washedwith Buffer C until the OD at 280 nm had returned to baseline indicatingthat no further protein was eluting from the column. Proteins wereeluted from the column using a linear gradient of sodium phosphate(10-400 mM sodium phosphate) in Buffer C. The fractions were tested forbinding to [³H] Inhibitor Compound as described in Example 1, and thefractions containing binding activity were pooled and dialyzed overnightagainst 2L Buffer A. The dialysate was applied to a MonoQ 5/5 column(Pharmacia) that had been equilibrated in Buffer A, the column waswashed with buffer A until the OD at 280 nm had returned to baselineindicating that no further protein was eluting from the column. Theproteins were then eluted using a linear gradient of sodium chloride(zero to 1 M) in Buffer A. Each fraction was tested for binding to [³H]Inhibitor Compound as above. Aliquots of the fractions containingbinding activity were applied to 4-20% or 4-12% polyacrylamide gels(Novex) and electrophoresis was performed in the presence of SDS. Thecompleted gels were stained with a silver stain kit according to themanufacturers instructions (Daiichi Pure Chemical Co. Ltd.). The twopeak binding fractions are referred to as “PKG purified fraction” insubsequent experiments.

[0116] In the peak fractions a 120 kDa protein was identified thateluted with the same profile as the [³H] Inhibitor Compound bindingactivity. This protein was excised from a Coomassie Blue stainedpolyacrylamide gel, tryptic peptides generated and peptide sequenceobtained. The peptides obtained are shown below: 1. AENRQFLA [SEQ IDNO:1] 2. VLYIL [SEQ ID NO:2] 3. LVSIK [SEQ ID NO:3] 4. EDTQAEDARLLGHLEK[SEQ ID NO:4] 5. EMPTASTGTPEQQQQQQQQ [SEQ ID NO:5] 6. HGEEQQQERKPSQQQQN[SEQ ID NO:6] 7. VFLXIV [SEQ ID NO:7]

EXAMPLE 4

[0117] Cloning of the cDNA Encoding the E. tenella Inhibitor CompoundBinding Protein

[0118] A series of degenerate oligonucleotides were synthesized from thesequence of the peptides listed above. Combinations of these degenerateoligonucleotides were used in coupled reverse transcriptase-polymerasechain reactions (RT-PCR). RNA prepared from unsporulated oocysts of E.tenella was used as substrate with random hexamer primers in a reactioncatalyzed by reverse transcriptase to synthesize cDNA copies of the RNA.An aliquot of this cDNA was then used as a template in a series of PCRreactions along with various combinations of the degenerateoligonucleotides described above.

[0119] Nested PCR reactions using the primary PCR reaction product astemplate and a different degenerate oligonucleotide(s) from the initialgroup of primers described above were performed to better identifyinteresting cDNA products. Those skilled in the method recognize thatpromiscuous hybridization of primers can and does occur during PCRreactions and that the frequency of promiscuous hybridization increasesenormously when degenerate oligonucleotides are used. By performing asecondary or nested PCR reaction with a different single or pair ofdegenerate oligonucleotide primers derived from the initial set ofpeptide sequences, one can have far greater confidence in theauthenticity of the PCR reaction product(s) as it relates to the proteinof interest. Using this criteria for selection, several of the RT-PCRreaction products were cloned and sequenced in an attempt to identify aninternal deduced peptide identical to one of the native peptides and/oran open reading frame (ORF) that bears resemblence to a known sequencein sequence databases. One of the RT-PCR products (clone Et.52035) has asingle open reading frame defined at one end by a degenerateoligonucleotide derived from the fourth peptide listed above and at theother end by a degenerate oligonucleotide derived from the first peptidelisted above. Moreover, this ORF from clone Et.52035 has limited aminoacid sequence identity with a D. melanogaster cGMP-dependent proteinkinase (PKG) and the regulatory subunit of a S. cerevisiaecAMP-dependent protein kinase. The nucleotide sequence of clone Et.52035is shown as SEQ ID NO: 8, and the deduced amino acid sequence is shownas SEQ ID NO: 9. The degenerate oligonucleotides that were used togenerate the Et.52035 PCR product are listed below. In the primary PCRreaction, oligonucleotides AG1-2 (from peptide SEQ ID NO: 5) and AG4-1(from SEQ ID NO: 1) were used as a primer pair. The product of this PCRwas then used as substrate in a secondary reaction with oligonucleotidesAG3-1 (from peptide SEQ ID NO: 4) and AG4-1 as a primer pair. AG1-2: 5′-ACN GGN CAN CCN GA(A/G)CA(A/G)CA -3′ AG4-1: 5′- GCN A(A/G)(A/G)AA(C/T)TGN C(T/G)(A/G)TT(C/T)TC-3′ AG3-1: 5′- GA(A/G)GA(C/T)ACNCA(A/G)GCN GA(A/G)GA(C/T)GC-3′

[0120] The insert from clone Et.52035 was used as a hybridization probeto screen an E. tenella cDNA library. The cDNA library was constructedby methods common in the art. Total RNA was prepared from unsporulatedoocysts (USO) of E. tenella. Poly (A) RNA was twice selected using oligo(dT) cellulose chromatography and used as a template for first strandcDNA synthesis with SuperScript reverse transcriptase (LifeTechnologies, Inc.) and primed with oligo (dT). Following second strandcDNA synthesis and EcoR I adapter ligation, double stranded cDNA wassize fractionated by column chromatography using Sephacryl S-500 HR andthen ligated into the phage vector lambda ZAP II (Stratagene). Ligationreaction products were then packaged using Gigapack III Gold packagingextract (Stratagene) according to the manufacturer's recommendations. Atotal of 1.25×10⁶ recombinant phage were screened using the 453 basepair insert from clone Et.52035. Several plaque-pure positive cloneswere isolated from each library and subcloned into pBluescript SK(Stratagene) by in vivo excision according to the manufacturer'srecommendations. Phagemid clones were characterized by restrictionenzyme mapping and partial nucleotide sequence analysis. Automated DNAsequence was performed using an Applied Biosystems model 373 instrumentwith the Prism FS cycle sequencing kit.

[0121] The longest cDNA clone purified in this screen (Et.PKG7) isnearly 4.3 kb in length with a deduced ORF of 1003 amino acids, capableof coding for a protein of 113 kDa. Each of the seven peptide sequencesfrom the biochemically purified 120 kDa E. tenella protein can belocated within this ORF. The nucleotide sequence of Et.PKG7 is shown asSEQ ID NO: 10, and the deduced amino acid sequence shown as is SEQ IDNO: 11. The deduced amino acid sequence still most closely resembles theDrosophila PKG; 31% identity to the dual cGMP binding domains and 45%identity to the catalytic domain. Unlike all other PKGs in which thesetwo functional domains are close to one another along the length of theprotein, the Eimeria gene product is predicted to have nearly 300 aminoacids between the two domains. This 300 amino acid length accounts forthe majority of the size difference between the Eimeria protein andother PKGs.

EXAMPLE 5

[0122] Cloning of the T. gondii cDNA Homologue of E. tenella PKG7

[0123] A 753 nucleotide length of E. tenella cDNA clone PKG7 (nucleotideposition 2573 to 3325 within SEQ ID NO: 10) was amplified by PCR andused as a probe to screen a T. gondii cDNA library using reducedhybridization stringency to isolate a PKG homologue from this relatedprotozoan parasite. This region of the Eimeria cDNA clone was chosenbecause it comprises the majority of the catalytic domain of the deducedprotein product and is the region which is most highly conservedevolutionarily among PKG entries in nucleic acid databases. Full lengthT. gondii cDNA clones nearly 4.0 kb in length have been sequenced andcode for a deduced protein 994 amino acids in length which is 69%identical and 77% similar to the protein predicted from E. tenella clonePKG7. The nucleotide sequence encoding T. gondii PKG is shown as SEQ IDNO: 12, and the deduced amino acid sequence as SEQ ID NO: 13.

EXAMPLE 6

[0124] Functional Recombinant Expression of the Protein Coded for by E.tenella PKG7

[0125] The open reading frame of cDNA clone Et.PKG7 has been modified byPCR to add six histidine residues at the C-terminal end of the protein.This has been accomplished using methods familiar to those skilled inthe art. Oligonucleotide primers which hybridize to the ends of thededuced open reading frame from clone Et.PKG7 were synthesized. The5′-oligonucleotide starts at what is presumed to be the initiatormethionine and has been modified to add a Bam HI cloning site. The3′-oligonucleotide carries sequence that is complementary to thenucleotide sequence that encodes the final six deduced amino acidresidues of Et PKG. Following this the synthetic oligonucleotide has 18nucleotides capable of coding for six consecutive histidine residues.This length in turn is followed by a TAA nucleotide triplet (atranslation termination codon) and then a Xba I restriction enzymerecognition sequence for cloning purposes. These two synthetic primerswere used in a PCR reaction with Et.PKG7 as template to generate a cDNAthat corresponds to the protein coding region and which can be subcloneddirectionally as a 5′-Bam HI/3′-Xba I fragment into the baculovirusexpression vector pFastBac I (Life Technologies) for recombinant proteinexpression in invertebrate cells. The hexa-histidine motif serves bothas an epitope to follow the expression and purification of therecombinant protein by Western blotting as well as an affinity tag toaid in the purification of recombinant Et.PKG7 by immobilized metalchelate affinity chromatography (IMAC). Western blot positiverecombinant protein expressed in invertebrate Sf9 cells has beenpartially purified by sequential IMAC and cGMP-agarose affinitychromatographies (see below) for biochemical characterization.

EXAMPLE 7

[0126] Identification of the Native 120 kDa E. tenella [ ³H] InhibitorCompound Binding Protein and the Recombinant Protein Expressed from cDNAClone Et.PKG7 as a cGMP-Dependent Protein Kinase

[0127] (a) PKG catalytic activity. E. tenella PKG purified fraction asdescribed above (0.05 μl) was incubated for 2 hours at room temperaturein 20 μl of an incubation mixture containing: 25 mM HEPES, pH 7.4, 10 mMMgCl₂, 5 mM beta-mercaptoethanol, 20 mM beta-glycerophosphate (or 100 μMsodium orthovanadate), 10 μM cGMP, 2 μM ATP, 10 nM [³³P] ATP (2000Ci/mmole), 400 μM Kemptide (or 2 mM Kemptide or 0.43 mg/ml myelin basicprotein). The reaction was then quenched with 2.5 μl of 0.25 Mphosphoric acid per 20 μl assay volume, and the mixture was spotted onWhatman P81 chromatography paper. The chromatography paper was allowedto dry for 2 minutes, washed 5 times for 3 min. each with 150 mls of 75mM phosphoric acid, then washed briefly with 95% ethanol. After airdrying, labeled kemptide remaining on the filter was quantitated byliquid scintillation counting.

[0128] The biochemically purified native E. tenella Inhibitor Compoundbinding protein contained very little kinase activity when tested in theabsence of cyclic nucleotide cofactors. Kinase activity was marginallystimulated when assayed in the presence of cAMP, but was stimulated bygreater than 400 fold with cGMP.

[0129] (b) “In-gel” kinase assay. To confirm that the kinase activitywas contributed by the 120 kDa polypeptide and not by another minorprotein in this biochemical fraction, an “in gel” kinase assay wasconducted (see method below). The in gel kinase assay was performedaccording to the manufacturers instructions (Stratagene In-Gel ProteinKinase Assay Kit, Instruction Manual Cat. # 206020, Revision # 117002)except that the substrate provided in the kit was replaced with 0.33 mgmyelin basic protein per ml of gel. In this method an SDS-polyacrylamidegel is impregnated with a kinase substrate and the purified fraction iselectrophoresed into the gel. Following extensive washing to renaturethe kinase after exposure to SDS, ³²P ATP and cGMP were added. The labelbecomes incorporated into the substrate at positions where catalyticallyactive kinases are present. In this assay label was incorporated only atthe position of the 120 kDa protein.

[0130] (c) Soluble proteins derived from a lysate of E. tenella werechromatographed on a HiLoad Q column (see Example 3 above). Fractionswere tested for kinase and binding activities. The results indicate thatthese two activities are coincident, providing further confirmation thatthe binding to [³H] Inhibitor Compound and kinase activity reside in thesame protein.

[0131] (d) cGMP agarose affinity chromatography. A 1 ml cGMP agarosecolumn (Biolog) was equilibrated with cGMP agarose buffer (50 mM Hepes,pH 7.4, 10% glycerol, 10 mM sodium fluoride, 0.1 mM sodiumorthovanadate, 1 mM EDTA) and the purified fraction applied to thecolumn. The column was washed with 10 ml of cGMP agarose buffer.Non-specifically bound proteins were removed by washing with 5 ml ofcGMP agarose buffer containing 1 mM GMP. Proteins were eluted with 5 mlof cGMP agarose buffer containing 15 mM cGMP and fractions werecollected. Fractions were analyzed for [³H]-Inhibitor Compound binding,PKG activity and by silver staining following polyacrylamide gelelectrophoresis. The affinity matrix, cGMP agarose (Biolog) binds cGMPbinding proteins. When the purified native parasite PKG fraction wasapplied to such a column all of the Inhibitor Compound binding activityand PKG catalytic activity bound to the column and could be eluted withcGMP but not with GMP (see below). Stained gels of the eluates showedthat the only protein eluted from the column was the 120 kDapolypeptide. Recombinant protein expressed in invertebrate cells fromcDNA clone Et. PKG7 and partially purified by IMAC chromatography alsobinds to the cGMP-agarose affinity matrix and can be specifically elutedwith buffer containing cGMP. Recombinant protein purified by thesesequential affinity chromatographies is also a catalytically active PKG.

[0132] The results from the above assays demonstrate that the 120 kDa E.tenella protein is a PKG and that it specifically binds InhibitorCompound. In addition, both the native and the recombinant expressed PKGcatalytic activity can be inhibited with Inhibitor Compound with an IC50of <5 nM.

1 16 1 8 PRT Artificial Sequence Inhibitor Binding Protein 1 Ala Glu AsnArg Gln Phe Leu Ala 1 5 2 5 PRT Artificial Sequence Inhibitor BindingProtein 2 Val Leu Tyr Ile Leu 1 5 3 5 PRT Artificial Sequence InhibitorBinding Protein 3 Leu Val Ser Ile Lys 1 5 4 16 PRT Artificial SequenceInhibitor Binding Protein 4 Glu Asp Thr Gln Ala Glu Asp Ala Arg Leu LeuGly His Leu Glu Lys 1 5 10 15 5 19 PRT Artificial Sequence InhibitorBinding Protein 5 Glu Met Pro Thr Ala Ser Thr Gly Thr Pro Glu Gln GlnGln Gln Gln 1 5 10 15 Gln Gln Gln 6 17 PRT Artificial Sequence InhibitorBinding Protein 6 His Gly Glu Glu Gln Gln Gln Glu Arg Lys Pro Ser GlnGln Gln Gln 1 5 10 15 Asn 7 6 PRT Artificial Sequence Inhibitor BindingProtein 7 Val Phe Leu Xaa Ile Val 1 5 8 453 DNA Artificial SequenceProbe 8 gaggataccc aggcggagga cgcgagacta ctgggtcacc tggagaagcgggagaaaact 60 ccttcggact tgtctttaat tcgggattct ctttcaacta atttagtttgttcttcttta 120 aatgacgcgg aagtggaggc tttggccaac gcggtggagt tcttcactttcaaaaaggga 180 gacgttgtca ccaaacaggg cgagagcggc agttatttct tcattgttcacagcggggag 240 tttgaggtga ttgtgaacga caaagtggtg aacaagattc taacgggccaggcctttggg 300 gaaatttctt taattcataa ttctgcaaga actgcaacaa tcaaaaccctcagcgaagat 360 gcagccttgt ggggcgtcca gagacaagtc ttcagggaaa ccctaaagcagctgagcagc 420 agaaactttg ccgagaaccg ccagttcctt gca 453 9 151 PRTArtificial Sequence PKG 9 Glu Asp Thr Gln Ala Glu Asp Ala Arg Leu LeuGly His Leu Glu Lys 1 5 10 15 Arg Glu Lys Thr Pro Ser Asp Leu Ser LeuIle Arg Asp Ser Leu Ser 20 25 30 Thr Asn Leu Val Cys Ser Ser Leu Asn AspAla Glu Val Glu Ala Leu 35 40 45 Ala Asn Ala Val Glu Phe Phe Thr Phe LysLys Gly Asp Val Val Thr 50 55 60 Lys Gln Gly Glu Ser Gly Ser Tyr Phe PheIle Val His Ser Gly Glu 65 70 75 80 Phe Glu Val Ile Val Asn Asp Lys ValVal Asn Lys Ile Leu Thr Gly 85 90 95 Gln Ala Phe Gly Glu Ile Ser Leu IleHis Asn Ser Ala Arg Thr Ala 100 105 110 Thr Ile Lys Thr Leu Ser Glu AspAla Ala Leu Trp Gly Val Gln Arg 115 120 125 Gln Val Phe Arg Glu Thr LeuLys Gln Leu Ser Ser Arg Asn Phe Ala 130 135 140 Glu Asn Arg Gln Phe LeuAla 145 150 10 4262 DNA Artificial Sequence PKG 10 ccagaggctg acgggtggttattgcttctc cgtttgagtc ccgcgcgcac ccttggatcg 60 tgctgtcttt cctttgcgctgcagcagcgc cgcgtgttac ggcagcagca gcagcagcag 120 cagcacaccc aacaacagcaggaatttgac gcttaattta gcgtggcacc ccagcagcag 180 gagcagcagc agtctagggcttcactgacg tgcgcgttca cagcagcagc agcagcagct 240 gcagcagcgg caacagcaacagtagtagta gcggcagcag cagctgcaga agcagcagcg 300 gcagcagcgt cagagtcagttgctgcctct tcctctctag ctgctgccca caatatgaat 360 gggatcagca gcagcaacagcaacagtagc agcagcagca gcagcagcag tgacagcccg 420 ccgtcggcag ttaatcatttgtagcatctc aagccacggg agcagcagca gcgcagcaac 480 agcagcagga gctccattccttgtatgggt gtggggaggc attctggtgg cccctctagc 540 tgctgctgca gcagcagcagcagcagcagc agcaacacgc gctgccttgc cgatcgcagc 600 agtttacact gaaaatgggcgcatgcagct ctaaggcgca gcaccagacc cgagatccgg 660 agccacgaga gcagcaggctgcgcaagaac agaaatcaac aggcccgagc ggcgctccta 720 acgacgcccc cgcacctgctgaggcggaga ggaagatgtc ggggtcaagc gccacagctc 780 ccaagggcga aatgcccacggccagtacgg gcaccccgga gcagcaacag cagcaacagc 840 agcagcagca gcagcagcaggaacagcagc agcaccccga gcatcagcag tcagagaagc 900 agcagcagca tggggaggagcagcagcaag agaggaaacc ctcgcagcag cagcaaaatg 960 aagaagcagc agcaccccacaagcacggtg gagagcggaa ggtccagaag gcaattaagc 1020 agcaggaaga cactcaagcagaagatgcga gactactggg tcacctggag aagcgggaga 1080 aaactccttc ggacttgtctttaattcggg attctctttc aactaattta gtttgttctt 1140 ctttaaatga cgcggaagtggaggctttgg ccaacgcggt ggagttcttc actttcaaaa 1200 agggagacgt tgtcaccaaacagggcgaga gcggcagtta tttcttcatt gttcacagcg 1260 gggagtttga ggtgattgtgaacgacaaag tggtgaacaa gattctaacg ggccaggcct 1320 ttggggaaat ttctttaattcataattctg caagaactgc aacaatcaaa accctcagcg 1380 aagatgcagc cttgtggggcgtccagagac aagtcttcag ggaaacccta aagcagctga 1440 gcagcagaaa ctttgccgagaaccgacagt tcttggcttc tgtgaaattc tttgaaatgt 1500 tgacggaggc ccagaagaatgtgataacca acgcgctggt ggtgcagtct ttccagcccg 1560 gccaggcaat tgttaaggaaggagaaaaag gagatgttct ttacatttta aaaagtggca 1620 aagccctcgt gtccatcaaaaacaaagaag tccgggttct ccagcgggga gaatactttg 1680 gcgagcgggc gctgctttatgatgaacctc gaagtgcaac aataacagca gaagagccga 1740 cagtttgtgt ctccattggcagagaccttt tggacagggt tttggggaat ctgcagcacg 1800 tgctcttccg caatattatgctcgaggcac tgcagcagag caaggtcttc gcctctttcc 1860 cgacggagca gctgagccgcttaattggct ctgtagtggt taaggactat cccgaaaatt 1920 atattatttt ggatcgagaaaaccgcacaa gggcgtcggc gtcggcgctg ttctcggcgc 1980 aaggcgtgcg tttcttcttcgtgttggagg gagaggtttc tgtgtttgcc tacaaagaca 2040 agtccagcag cagcagcagcagcagcagca gcagcagcag cagcagcagc gcggaggggg 2100 aaatggagct gcacctcatagataccctaa aaaggggaca agcttttgga gacgaatatg 2160 ttttgtctcc caacaaaccctttgcgcatt gcgtcaagag caacgggccg acgaagctcg 2220 cgctgctgac ggctagcgcgctgaccgcca ctttgggggg acaagacatt gacgaaaccc 2280 tcgactataa caacaaattagcgatcacga aaaagatgta tatttttagg tatttgtcgg 2340 agcagcaaac gcaaaccctaatcaaggcct tcaagaccgt cagatacact cagggggaat 2400 ccatcattcg ggaaggcgaaatagggtctc ggttcttcat catcaagctt ggagaagtgg 2460 tgattctgaa gggcgaaaagcgggtgcgca cgctgggccg tcacgactac ttcggcgaga 2520 gggctttgct gcacgacgagcggcggtctg caacagtagc agcaaacagt cccgaggtgg 2580 atctgtgggt tgtcgacaaagatgttttcc tccaaatcgt caaagggccc atgcttaccc 2640 acttggagga gcgcattcgcatgcaagaca ccaaagttga attcaaagac ttgaatgttg 2700 tccgagtggt cggcagagggacgttcggga ctgtaaagct ggtgcagcac attcccacgc 2760 aaatgcgcta cgccttaaaatgcgtttcga gaaaaagtgt tgtggcttta aatcaacaag 2820 accacattcg actagaaagagagataatgg cagaaaacga ccaccccttc atcattcgcc 2880 tggtgcggac attccgggataaggagtttc tgtatttctt gacggagctg gtgacgggtg 2940 gagagctgta cgacgctattcggaagttgg gtcttttagg gaggtaccag gcgcagtttt 3000 acttggcctc gatcgtgctggccatcgagt acctgcacga gcggaacatc gcgtaccggg 3060 acttgaagcc ggagaacattttgctggatt ctcagggata cgtcaaactc atcgacttcg 3120 gctgcgccaa aaaaatgcagggaagagcct acacgctcgt gggaactccg cactacatgg 3180 cgccggaggt cattttgggaaaaggatata ctctaacagc agacacttgg gcctttgggg 3240 tttgtcttta cgaattcatgtgcggccctt tgccctttgg aaacgacgcg gaggaccagc 3300 tcgagatctt cagagacattctcgcaggca aactcatatt cccccactac gtgactgacc 3360 aagacgccat aaaccttatgaagcggctgc tgtgccgttt gcctgaagtg cggattggct 3420 gctcaattaa tggatacaaagacattaagg agcacgcctt cttctcggac ttcgactggg 3480 acagacttgc agggagagatttgtctcctc cgcttttgcc taagggagaa atctacgcag 3540 aagacgcgga ggagggaggattggatattg aggaagacga gggcatagaa cttgaagacg 3600 aatatgaatg ggacaaggacttctaaaccc taaaccctag caaaccctaa accctcgttt 3660 tggtgcttta gcctcttcgggcttttctcc ctaaccgatt taccaagctg ccattgtgca 3720 gcagcagtga gggtcttcgcaatattttcg tttggaaatt tgctgctgct gctgcagcag 3780 cagcagcagc ggcagtggcggcagcagcag cggcagccca ctctgtgcga cgagagctgt 3840 tgctgctgtg ggtgcgctgctgctctcgct gctgccgttg cagcaccgca gcagcagcag 3900 cagcagcagc ggcagcagcagcagcggcag cagcagcagc agcgtgtctg actgtttttg 3960 ctgctattct gtaatcatattttgttggtt ctttagtgtt tgcaccttaa actcttgccg 4020 ttggctttgc tgctgcagtgctgctgcagc ggcggtttgc tgatgctgct gcagcggcgg 4080 tttgctgatg ctgctgcagcggcggtttgc tgctgctgct gctgctgctg cagcgcgaag 4140 ccagacttga gcccgcgtagggtctttccg attgctgcag ctgtcgacgc agagtctctt 4200 tattgtttta tataaattattaaacaaaaa taaaaaaata aaaataaaaa aaaaaaaaaa 4260 aa 4262 11 1003 PRTArtificial Sequence PKG 11 Met Gly Ala Cys Ser Ser Lys Ala Gln His GlnThr Arg Asp Pro Glu 1 5 10 15 Pro Arg Glu Gln Gln Ala Ala Gln Glu GlnLys Ser Thr Gly Pro Ser 20 25 30 Gly Ala Pro Asn Asp Ala Pro Ala Pro AlaGlu Ala Glu Arg Lys Met 35 40 45 Ser Gly Ser Ser Ala Thr Ala Pro Lys GlyGlu Met Pro Thr Ala Ser 50 55 60 Thr Gly Thr Pro Glu Gln Gln Gln Gln GlnGln Gln Gln Gln Gln Gln 65 70 75 80 Gln Gln Glu Gln Gln Gln His Pro GluHis Gln Gln Ser Glu Lys Gln 85 90 95 Gln Gln His Gly Glu Glu Gln Gln GlnGlu Arg Lys Pro Ser Gln Gln 100 105 110 Gln Gln Asn Glu Glu Ala Ala AlaPro His Lys His Gly Gly Glu Arg 115 120 125 Lys Val Gln Lys Ala Ile LysGln Gln Glu Asp Thr Gln Ala Glu Asp 130 135 140 Ala Arg Leu Leu Gly HisLeu Glu Lys Arg Glu Lys Thr Pro Ser Asp 145 150 155 160 Leu Ser Leu IleArg Asp Ser Leu Ser Thr Asn Leu Val Cys Ser Ser 165 170 175 Leu Asn AspAla Glu Val Glu Ala Leu Ala Asn Ala Val Glu Phe Phe 180 185 190 Thr PheLys Lys Gly Asp Val Val Thr Lys Gln Gly Glu Ser Gly Ser 195 200 205 TyrPhe Phe Ile Val His Ser Gly Glu Phe Glu Val Ile Val Asn Asp 210 215 220Lys Val Val Asn Lys Ile Leu Thr Gly Gln Ala Phe Gly Glu Ile Ser 225 230235 240 Leu Ile His Asn Ser Ala Arg Thr Ala Thr Ile Lys Thr Leu Ser Glu245 250 255 Asp Ala Ala Leu Trp Gly Val Gln Arg Gln Val Phe Arg Glu ThrLeu 260 265 270 Lys Gln Leu Ser Ser Arg Asn Phe Ala Glu Asn Arg Gln PheLeu Ala 275 280 285 Ser Val Lys Phe Phe Glu Met Leu Thr Glu Ala Gln LysAsn Val Ile 290 295 300 Thr Asn Ala Leu Val Val Gln Ser Phe Gln Pro GlyGln Ala Ile Val 305 310 315 320 Lys Glu Gly Glu Lys Gly Asp Val Leu TyrIle Leu Lys Ser Gly Lys 325 330 335 Ala Leu Val Ser Ile Lys Asn Lys GluVal Arg Val Leu Gln Arg Gly 340 345 350 Glu Tyr Phe Gly Glu Arg Ala LeuLeu Tyr Asp Glu Pro Arg Ser Ala 355 360 365 Thr Ile Thr Ala Glu Glu ProThr Val Cys Val Ser Ile Gly Arg Asp 370 375 380 Leu Leu Asp Arg Val LeuGly Asn Leu Gln His Val Leu Phe Arg Asn 385 390 395 400 Ile Met Leu GluAla Leu Gln Gln Ser Lys Val Phe Ala Ser Phe Pro 405 410 415 Thr Glu GlnLeu Ser Arg Leu Ile Gly Ser Val Val Val Lys Asp Tyr 420 425 430 Pro GluAsn Tyr Ile Ile Leu Asp Arg Glu Asn Arg Thr Arg Ala Ser 435 440 445 AlaSer Ala Leu Phe Ser Ala Gln Gly Val Arg Phe Phe Phe Val Leu 450 455 460Glu Gly Glu Val Ser Val Phe Ala Tyr Lys Asp Lys Ser Ser Ser Ser 465 470475 480 Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ala Glu Gly Glu485 490 495 Met Glu Leu His Leu Ile Asp Thr Leu Lys Arg Gly Gln Ala PheGly 500 505 510 Asp Glu Tyr Val Leu Ser Pro Asn Lys Pro Phe Ala His CysVal Lys 515 520 525 Ser Asn Gly Pro Thr Lys Leu Ala Leu Leu Thr Ala SerAla Leu Thr 530 535 540 Ala Thr Leu Gly Gly Gln Asp Ile Asp Glu Thr LeuAsp Tyr Asn Asn 545 550 555 560 Lys Leu Ala Ile Thr Lys Lys Met Tyr IlePhe Arg Tyr Leu Ser Glu 565 570 575 Gln Gln Thr Gln Thr Leu Ile Lys AlaPhe Lys Thr Val Arg Tyr Thr 580 585 590 Gln Gly Glu Ser Ile Ile Arg GluGly Glu Ile Gly Ser Arg Phe Phe 595 600 605 Ile Ile Lys Leu Gly Glu ValVal Ile Leu Lys Gly Glu Lys Arg Val 610 615 620 Arg Thr Leu Gly Arg HisAsp Tyr Phe Gly Glu Arg Ala Leu Leu His 625 630 635 640 Asp Glu Arg ArgSer Ala Thr Val Ala Ala Asn Ser Pro Glu Val Asp 645 650 655 Leu Trp ValVal Asp Lys Asp Val Phe Leu Gln Ile Val Lys Gly Pro 660 665 670 Met LeuThr His Leu Glu Glu Arg Ile Arg Met Gln Asp Thr Lys Val 675 680 685 GluPhe Lys Asp Leu Asn Val Val Arg Val Val Gly Arg Gly Thr Phe 690 695 700Gly Thr Val Lys Leu Val Gln His Ile Pro Thr Gln Met Arg Tyr Ala 705 710715 720 Leu Lys Cys Val Ser Arg Lys Ser Val Val Ala Leu Asn Gln Gln Asp725 730 735 His Ile Arg Leu Glu Arg Glu Ile Met Ala Glu Asn Asp His ProPhe 740 745 750 Ile Ile Arg Leu Val Arg Thr Phe Arg Asp Lys Glu Phe LeuTyr Phe 755 760 765 Leu Thr Glu Leu Val Thr Gly Gly Glu Leu Tyr Asp AlaIle Arg Lys 770 775 780 Leu Gly Leu Leu Gly Arg Tyr Gln Ala Gln Phe TyrLeu Ala Ser Ile 785 790 795 800 Val Leu Ala Ile Glu Tyr Leu His Glu ArgAsn Ile Ala Tyr Arg Asp 805 810 815 Leu Lys Pro Glu Asn Ile Leu Leu AspSer Gln Gly Tyr Val Lys Leu 820 825 830 Ile Asp Phe Gly Cys Ala Lys LysMet Gln Gly Arg Ala Tyr Thr Leu 835 840 845 Val Gly Thr Pro His Tyr MetAla Pro Glu Val Ile Leu Gly Lys Gly 850 855 860 Tyr Thr Leu Thr Ala AspThr Trp Ala Phe Gly Val Cys Leu Tyr Glu 865 870 875 880 Phe Met Cys GlyPro Leu Pro Phe Gly Asn Asp Ala Glu Asp Gln Leu 885 890 895 Glu Ile PheArg Asp Ile Leu Ala Gly Lys Leu Ile Phe Pro His Tyr 900 905 910 Val ThrAsp Gln Asp Ala Ile Asn Leu Met Lys Arg Leu Leu Cys Arg 915 920 925 LeuPro Glu Val Arg Ile Gly Cys Ser Ile Asn Gly Tyr Lys Asp Ile 930 935 940Lys Glu His Ala Phe Phe Ser Asp Phe Asp Trp Asp Arg Leu Ala Gly 945 950955 960 Arg Asp Leu Ser Pro Pro Leu Leu Pro Lys Gly Glu Ile Tyr Ala Glu965 970 975 Asp Ala Glu Glu Gly Gly Leu Asp Ile Glu Glu Asp Glu Gly IleGlu 980 985 990 Leu Glu Asp Glu Tyr Glu Trp Asp Lys Asp Phe 995 1000 127796 DNA Artificial Sequence PKG 12 cgagatcgcc caagacaggg acgagtttctcccgttcagc agagcggaga gctctagcgg 60 gttctgtccc tgctcaaaga gggcaagtcgtctcgcctct aacgcttgaa cgcagcgcgt 120 ctgggcgact ctccgagcga ctgctcccgtttgcgaactt gcgtcgcgca gacccgctga 180 gaggctcgct gacgagggca gtctttctaccacgttttcg tgtgctttca agagtgccgt 240 ctgtggtcga cagaaagatg gtgcaaaagcacacgaaagt tctcacggca gacaccagct 300 gcggtaatcg ctctcaaccg acacaggctcgtcgtttccg gctgtacagg cgccattagc 360 gagagttggc tgtgtccgag cagcaaaggccgacatgtcc ttttctcgct tctccccgtt 420 cttttccccc tttccgtgct tcctgtattcaaaagagcga agaggggcaa gaaaaggggg 480 aaaggcacga aggacataag tcgatcaacagaaaataatc cctccctgga ctcgctgctg 540 gtcccgcgcc agctagttgt cttttattagggagggacct gagcgacgac cagggcgcgg 600 tcgccgtctc acgttctgcg cttcctgtcggaatcgtttg ccagcgtttc agcggcagag 660 tgcaagacgc gaaggacagc cttagcaaacggtcgcaaag cactccaccg tttccgccct 720 cgcggtgtct aaacactgcc gtctacgctggtgaggtggc aaaggcggga gcgccacaga 780 tttgtgacgg cagatgcgac cctgctttcttcttccttcg tcttctttct gctccgcaca 840 cctcgccact ggacgaaaga agaaggaagcagaagaaaga cgaggcgtgt ggagcggtga 900 ttgccgtctc ttcgcgcttc ttttcctctgcctcaggctc cttgtctgca aacggcagag 960 aagcgcgaag aaaaggagac ggagtccgaggaacagacgt cgagcacaca ccttttcttt 1020 cgtgtcgctt ccccggcttt cgtttctcccgctcgtgtgt ggaaaagaaa gcacagcgaa 1080 ggggccgaaa gcaaagaggg tcgcactcgtcccctctctc tctctctgct ccgtgttgca 1140 cttggcggct agcgtgagca ggggagagagagagagacga ggcacaacgt gaaccgccga 1200 tcggcgctcg actctcgtct ctccccgtctgctgcgctca cgtggcacct agccgcgagc 1260 tgagagcaga gaggggcaga cgacgcgagtgcaccgtgga cgcgcgctct ctgactggga 1320 actgacgggc gcgagccagc agcgactgccgcgcgcgaga gactgaccct tgactgcccg 1380 cgctcggtcg tcgctgacgg acaagcctctcggcttctag gcgagaggac accgcgtacc 1440 agcggtcctc tgttcggaga gccgaagatccgctctcctg tggcgcatgg tcgccaggag 1500 gttgtctctc ctcgcgcgaa aatgggcgcttgcatttcca aaaatagttc caacagagag 1560 gagcgcgctt ttacccgcga acgtaaaggtttttatcaag ggctcgcgtt tcacggtctt 1620 ctgctctgtc cgcctcgaaa caaacagttgccgagcgcaa agtgccagaa gacgagacag 1680 gcggagcttt gtttgtcaac cggccagtgcaccgccagga gcggcaggcg acgagacctc 1740 tgcgacgggt gccggtcacg tggcggtcctcgccgtccgc tgctctggag acgctgccca 1800 gctgccgaag aagcttcgag gaactcccttgccagggtag acggaacgcg cgacggcttc 1860 ttcgaagctc cttgagggaa cggtcccatctgccttgcgc agcctccgca gcggagttgg 1920 agagagctcc ggacggtgtg tgcccagacctcggaggcgt cgcctcaacc tctctcgagg 1980 cctgccacac acgggtctgg gcgaggaaccggggacggcg aacgccgagc aaggaggcgt 2040 gacggagaag cgctccttgg cccctgccgcttgcggctcg ttcctccgca ctgcctcttc 2100 aaagacacta gagagacact ggcagggatgaactctccca agactctgga tttctgtgat 2160 ctctctgtga ccgtccctac ttgagagggttctgagacct ggcggaggcg caagaggacg 2220 accccaagcg cgaagcgccg aaccaggacgccgcctccgc gttctcctgc tggggttcgc 2280 gcttcgcggc ttggtcctgc ttccctctgaggctccagaa ggcccgaaag aaaaacccgg 2340 cggagaccgg aagggagact ccgaggtcttccgggctttc tttttgggcc gcctctggcc 2400 aaaccggcgc agaaggcgat tctgaagcaagatgactcgc acacggagga tttggccgcg 2460 tcttccgcta agacttcgtt ctactgagcgtgtgcctcct agagaaactc aacgcgcacc 2520 tggcatatcg cgagaagacg cctgcagacttctctttgag ttgcgcgtgg accgtatagc 2580 gctcttctgc ggacgtctga ttgcgctgatccaagacagc ctgaaggcga atcttgtctg 2640 ctcctctctg aacgcgacta ggttctgtcggacttccgct tagaacagac gaggagagac 2700 aatgagggcg aaatcgacgc tctcgcagtcgcgatgcagt tcttcacctt ttactcccgc 2760 tttagctgcg agagcgtcag cgctacgtcaagaagtggaa caagaaaggc gacgtggtga 2820 ccaagcaggg cgaacctgga agctacttttgttctttccg ctgcaccact ggttcgtccc 2880 gcttggacct tcgatgaaaa tcatcattcacagcgggacg tttgacgtgc tcgtcaacga 2940 caagcgcgtt agtagtaagt gtcgccctgcaaactgcacg agcagttgct gttcgcgcaa 3000 aatgcgatgg acaagggaaa agcctttggagaaatcgcgc tcattcacaa ttacgctacc 3060 tgttcccttt tcggaaacct ctttagcgcgagtaagtgtt taccgaaaga tctgcgacgg 3120 ttgtcgcaag ctccacagaa ggtgccctctatggctttct agacgctgcc aacagcgttc 3180 gaggtgtctt ccacgggaga ggggtgtccagcgtcacact ttcagagaaa cgctgaagca 3240 gctgagcagc ccccacaggt cgcagtgtgaaagtctcttt gcgacttcgt cgactcgtcg 3300 cgaaactttg cggagaacag acagtttttggcctccgtca agttcttcga gctttgaaac 3360 gcctcttgtc tgtcaaaaac cggaggcagttcaagaagct aatgttgaca gaggcgcaga 3420 aaaacgtcat caccaatgcg ctggtcgtggttacaactgt ctccgcgtct ttttgcagta 3480 gtggttacgc gaccagcacc agaacttcaagcctggacag cccattgtga aggaaggcga 3540 cgcaggagac tcttgaagtt cggacctgtcgggtaacact tccttccgct gcgtcctctg 3600 gtcctctaca tcttgaagag cggcaaagcgaaggtctcca tcggcggacg caggagatgt 3660 agaacttctc gccgtttcgc ttccagaggtagccgcctgc ggagatccgc atgctgcgga 3720 aaggcgacta cttcggagag cgcgcgttgccctctaggcg tacgacgcct ttccgctgat 3780 gaagcctctc gcgcgcaacg tgtacaaagagccgaggagc gcaaccatca cggcagaaga 3840 gttcaccgtc acatgtttct cggctcctcgcgttggtagt gccgtcttct caagtggcag 3900 tgcgtgtcga tcggccgcga gctgctggatcgcgtcttgg gaaatctgca acgcacagct 3960 agccggcgct cgacgaccta gcgcagaaccctttagacgt gcacgttctg ttcagaaaca 4020 tcatggtcga ggctctgcag caaagcaaagcgtgcaagac aagtctttgt agtaccagct 4080 ccgagacgtc gtttcgtttc tctacgaattgtttcaaggc gaccagctga gcaaattgat 4140 cgaagcggcc agatgcttaa caaagttccgctggtcgact cgtttaacta gcttcgccgg 4200 gtggtgaagg actacggcgc agactacgtcattctggata aggaaaacaa caccacttcc 4260 tgatgccgcg tctgatgcag taagacctattccttttgtt gacgaaggga attcgcttct 4320 tctttgtcct cgaaggagag ctgtcggtgtctgcttccct taagcgaaga agaaacagga 4380 gcttcctctc gacagccaca acgcctacacacaaaaccca gcgacgaaag aggaagaacg 4440 gaagctcgca tgcggatgtg tgttttgggtcgctgctttc tccttcttgc cttcgagcgt 4500 gcgactctga aacgcggcca agcattcggtgaagaatacg tccttaatcc cgctgagact 4560 ttgcgccggt tcgtaagcca cttcttatgcaggaattagg cactcgacct ttcaaccact 4620 atgtcaaaag tgtcggccct tgcaagctcggtgagctgga aagttggtga tacagttttc 4680 acagccggga acgttcgagc ctctgtttacttcgtccgtg ttgacggcga ctttgggagg 4740 cgaagacatc gagacaaatg aagcaggcacaactgccgct gaaaccctcc gcttctgtag 4800 gatgagacgc tggacttcaa taacaaacgcgcgattattc ggaagatgta ctactctgcg 4860 acctgaagtt attgtttgcg cgctaataagccttctacat cattttcaga tatctgtcag 4920 atcaccaaat gacgatgctc atcaaagcttgtaaaagtct atagacagtc tagtggttta 4980 ctgctacgag tagtttcgaa tcaaaactgtcagatacatg tcgggagagt acatcatcaa 5040 agaaggcgaa agttttgaca gtctatgtacagccctctca tgtagtagtt tcttccgctt 5100 cgcggcacac ggttcttcat catcaaagccggtgaagttg cgattctgaa gcgccgtgtg 5160 ccaagaagta gtagtttcgg ccacttcaacgctaagactt gaacaacaag cgcctccgca 5220 ccctcggccg ccacgactat tttggggaaccttgttgttc gcggaggcgt gggagccggc 5280 ggtgctgata aaaccccttg gggcgttgctgtacgataag cctaggacgg cctctgtgtg 5340 tgccaattct cccgcaacga catgctattcggatcctgcc ggagacacac acggttaaga 5400 gcgggggtcg acctctgggt cgtcgataagtcggttttca acgaaatcat cgcccccagc 5460 tggagaccca gcagctattc agccaaaagttgctttagta caaggggcct atgcttgctc 5520 acttggagga aagaatccgc atgcaagacagttccccgga tacgaacgag tgaacctcct 5580 ttcttaggcg tacgttctgt ccaaggtcgagttccaggac ttgcaagtgg tcagagtggt 5640 cggcagagga ggttccagct caaggtcctgaacgttcacc agtctcacca gccgtctcct 5700 accttcggca ccgttaagct cgtgcgccatgtgccgacag atattcgcta tggaagccgt 5760 ggcaattcga gcacgcggta cacggctgtctataagcgat tgcgctcaag tgtgtctcaa 5820 ggcgaagcgt catcgctctc agtcagcaacacgcgagttc acacagagtt ccgcttcgca 5880 gtagcgagag tcagtcgttg aacacattcgcctggaacgc gaaatcatgg cggaaaacga 5940 ccatcctttc ttgtgtaagc ggaccttgcgctttagtacc gccttttgct ggtaggaaag 6000 atcattcggc tagtgagaac gttccgcgacaaggagttcc tatacttcct tagtaagccg 6060 atcactcttg caaggcgctg ttcctcaaggatatgaagga cacggaactc gtcacaggag 6120 gcgaactgta cgatgccatc cggaagctaggtgccttgag cagtgtcctc cgcttgacat 6180 gctacggtag gccttcgatc gactgcttgctcggtcgcag gcccagttct acctcgcctc 6240 cattgtcctc ctgacgaacg agccagcgtccgggtcaaga tggagcggag gtaacaggag 6300 gccatcgagt atctccacga acgaaacatcgcgtacaggg acttgaagcc cggtagctca 6360 tagaggtgct tgctttgtag cgcatgtccctgaacttcgg tgagaacatt ctcctcgaca 6420 gccaaggcta cgtcaaactc atcgatttcgactcttgtaa gaggagctgt cggttccgat 6480 gcagtttgag tagctaaagc gctgtgcgaaaaagatgcaa ggtcgcgcct acacccttgt 6540 cggcacacct cgacacgctt tttctacgttccagcgcgga tgtgggaaca gccgtgtgga 6600 cactacatgg ctcctgaagt cattcttggcaaggggtaca cgctgacggc gtgatgtacc 6660 gaggacttca gtaagaaccg ttccccatgtgcgactgccg agacacttgg gcgttcggcg 6720 tctgcctgta cgagttcatg tgcggacctctctgtgaacc cgcaagccgc agacggacat 6780 gctcaagtac acgcctggag tccccttcggaaacgatgct gaagatcaac tggaaatctt 6840 ccgagacatt aggggaagcc tttgctacgacttctagttg acctttagaa ggctctgtaa 6900 ctcacaggaa aactcgtttt cccgcactatgtcaccgacc aagatgcgat gagtgtcctt 6960 ttgagcaaaa gggcgtgata cagtggctggttctacgcta caacctgatg aagaggcttt 7020 tgtgtcgtct ccctgaggtc cgaatcggatgttggactac ttctccgaaa acacagcaga 7080 gggactccag gcttagccta gctccatcaacggctacaag gacatcaagg agcacgcatt 7140 tttcggagac cgaggtagtt gccgatgttcctgtagttcc tcgtgcgtaa aaagcctctg 7200 ttcgactggg acaaactggc aggtcgtggcttgccgccac ccctcgcacc aagctgaccc 7260 tgtttgaccg tccagcaccg aacggcggtggggagcgtgg gaaaggcgaa acctacgcag 7320 aagatactga acaatcctct ttcgagctggctttccgctt tggatgcgtc ttctatgact 7380 tgttaggaga aagctcgacc acgaggatgacacgatcgtt ttggaagacg agtatgactg 7440 ggacaaggat tgctcctact gtgctagcaaaaccttctgc tcatactgac cctgttccta 7500 ttctgatttt tcagcttagg tgtttgttccccgtcagtgc tgaaagtgcc aagactaaaa 7560 agtcgaatcc acaaacaagg ggcagtcacgactttcacgg gctccttctc tcttctgact 7620 cttctgtgga cctgcaactt cctgcatgaccgaggaagag agaagactga gaagacacct 7680 ggacgttgaa ggacgtactg gaaacgatccgtgcataagc atgagcgcga aaaaaaaaaa 7740 aaaaaaaact ttgctaggca cgtattcgtactcgcgcttt tttttttttt tttttt 7796 13 994 PRT Artificial Sequence PKG 13Met Gly Ala Cys Ile Ser Lys Asn Ser Ser Ala Arg Val Ser Arg Ser 1 5 1015 Ser Ala Leu Ser Ala Ser Lys Gln Thr Val Ala Ala Ser Ala Pro Pro 20 2530 Gly Ala Ala Gly Asp Glu Thr Ser Ala Thr Gly Ala Ala Glu Glu Ala 35 4045 Ser Arg Asn Ser Leu Ala Arg Val Asp Gly Thr Arg Ala Ser Ala Ala 50 5560 Glu Leu Glu Arg Ala Pro Asp Gly Val Cys Pro Asp Arg Glu Glu Pro 65 7075 80 Gly Thr Ala Asn Ala Glu Gln Gly Gly Val Thr Glu Lys Lys Asp Thr 8590 95 Arg Glu Thr Leu Ala Gly Met Asn Ser Pro Lys Thr Leu Glu Ala Glu100 105 110 Ala Gln Glu Asp Asp Leu Lys Arg Glu Ala Pro Asn Gln Asp ValPro 115 120 125 Ser Glu Ala Pro Glu Gly Pro Lys Glu Lys Pro Gly Gly AspArg Lys 130 135 140 Pro Ala Gln Lys Ala Ile Leu Lys Gln Asp Asp Ser HisThr Glu Glu 145 150 155 160 Glu Lys Leu Asn Ala His Leu Ala Tyr Arg GluLys Thr Pro Ala Asp 165 170 175 Phe Ala Leu Ile Gln Asp Ser Leu Lys AlaAsn Leu Val Cys Ser Ser 180 185 190 Leu Asn Glu Gly Glu Ile Asp Ala LeuAla Val Ala Met Gln Phe Phe 195 200 205 Thr Phe Lys Lys Gly Asp Val ValThr Lys Gln Gly Glu Pro Gly Ser 210 215 220 Tyr Phe Phe Ile Ile His SerGly Thr Phe Asp Val Leu Val Asn Asp 225 230 235 240 Lys Arg Val Asn AlaMet Asp Lys Gly Lys Ala Phe Gly Glu Ile Ala 245 250 255 Leu Ile His AsnThr Glu Arg Ser Ala Thr Val Val Ala Ser Ser Thr 260 265 270 Glu Gly AlaLeu Trp Gly Val Gln Arg His Thr Phe Arg Glu Thr Leu 275 280 285 Lys GlnLeu Ser Ser Arg Asn Phe Ala Glu Asn Arg Gln Phe Leu Ala 290 295 300 SerVal Lys Phe Phe Glu Met Leu Thr Glu Ala Gln Lys Asn Val Ile 305 310 315320 Thr Asn Ala Leu Val Val Glu Asn Phe Lys Pro Gly Gln Pro Ile Val 325330 335 Lys Glu Gly Asp Ala Gly Asp Val Leu Tyr Ile Leu Lys Ser Gly Lys340 345 350 Ala Lys Val Ser Ile Gly Gly Arg Glu Ile Arg Met Leu Arg LysGly 355 360 365 Asp Tyr Phe Gly Glu Arg Ala Leu Leu Tyr Lys Glu Pro ArgSer Ala 370 375 380 Thr Ile Thr Ala Glu Glu Phe Thr Val Cys Val Ser IleGly Arg Glu 385 390 395 400 Leu Leu Asp Arg Val Leu Gly Asn Leu Gln HisVal Leu Phe Arg Asn 405 410 415 Ile Met Val Glu Ala Leu Gln Gln Ser LysVal Tyr Glu Leu Phe Gln 420 425 430 Gly Asp Gln Leu Ser Lys Leu Ile GluAla Ala Val Val Lys Asp Tyr 435 440 445 Gly Ala Asp Tyr Val Ile Leu AspLys Glu Asn Lys Thr Lys Gly Ile 450 455 460 Arg Phe Phe Phe Val Leu GluGly Glu Leu Ser Val Tyr Ala Tyr Thr 465 470 475 480 Gln Asn Pro Ala ThrLys Glu Glu Glu Arg Lys Leu Ala Ala Thr Leu 485 490 495 Lys Arg Gly GlnAla Phe Gly Glu Glu Tyr Val Leu Asn Pro Thr Arg 500 505 510 Pro Phe AsnHis Tyr Val Lys Ser Val Gly Pro Cys Lys Leu Ala Leu 515 520 525 Phe ThrSer Ser Val Leu Thr Ala Thr Leu Gly Gly Glu Asp Ile Asp 530 535 540 GluThr Leu Asp Phe Asn Asn Lys Arg Ala Ile Ile Arg Lys Met Tyr 545 550 555560 Ile Phe Arg Tyr Leu Ser Asp His Gln Met Thr Met Leu Ile Lys Ala 565570 575 Phe Lys Thr Val Arg Tyr Met Ser Gly Glu Tyr Ile Ile Lys Glu Gly580 585 590 Glu Arg Gly Thr Arg Phe Phe Ile Ile Lys Ala Gly Glu Val AlaIle 595 600 605 Leu Lys Asn Asn Lys Arg Leu Arg Thr Leu Gly Arg His AspTyr Phe 610 615 620 Gly Glu Arg Ala Leu Leu Tyr Asp Glu Pro Arg Thr AlaSer Val Cys 625 630 635 640 Ala Asn Ser Ala Gly Val Asp Leu Trp Val ValAsp Lys Ser Val Phe 645 650 655 Asn Glu Ile Ile Lys Gly Pro Met Leu AlaHis Leu Glu Glu Arg Ile 660 665 670 Arg Met Gln Asp Thr Lys Val Glu PheGln Asp Leu Gln Val Val Arg 675 680 685 Val Val Gly Arg Gly Thr Phe GlyThr Val Lys Leu Val Arg His Val 690 695 700 Pro Thr Asp Ile Arg Tyr AlaLeu Lys Cys Val Ser Arg Arg Ser Val 705 710 715 720 Ile Ala Leu Ser GlnGln Gln His Ile Arg Leu Glu Arg Glu Ile Met 725 730 735 Ala Glu Asn AspHis Pro Phe Ile Ile Arg Leu Val Arg Thr Phe Arg 740 745 750 Asp Lys GluPhe Leu Tyr Phe Leu Thr Glu Leu Val Thr Gly Gly Glu 755 760 765 Leu TyrAsp Ala Ile Arg Lys Leu Gly Leu Leu Ala Arg Ser Gln Ala 770 775 780 GlnPhe Tyr Leu Ala Ser Ile Val Leu Ala Ile Glu Tyr Leu His Glu 785 790 795800 Arg Asn Ile Ala Tyr Arg Asp Leu Lys Pro Glu Asn Ile Leu Leu Asp 805810 815 Ser Gln Gly Tyr Val Lys Leu Ile Asp Phe Gly Cys Ala Lys Lys Met820 825 830 Gln Gly Arg Ala Tyr Thr Leu Val Gly Thr Pro His Tyr Met AlaPro 835 840 845 Glu Val Ile Leu Gly Lys Gly Tyr Thr Leu Thr Ala Asp ThrTrp Ala 850 855 860 Phe Gly Val Cys Leu Tyr Glu Phe Met Cys Gly Pro LeuPro Phe Gly 865 870 875 880 Asn Asp Ala Glu Asp Gln Leu Glu Ile Phe ArgAsp Ile Leu Thr Gly 885 890 895 Lys Leu Val Phe Pro His Tyr Val Thr AspGln Asp Ala Ile Asn Leu 900 905 910 Met Lys Arg Leu Leu Cys Arg Leu ProGlu Val Arg Ile Gly Cys Ser 915 920 925 Ile Asn Gly Tyr Lys Asp Ile LysGlu His Ala Phe Phe Gly Asp Phe 930 935 940 Asp Trp Asp Lys Leu Ala GlyArg Gly Leu Pro Pro Pro Leu Ala Pro 945 950 955 960 Lys Gly Glu Thr TyrAla Glu Asp Thr Glu Gln Ser Ser Phe Glu Leu 965 970 975 Asp Glu Asp AspThr Ile Val Leu Glu Asp Glu Tyr Asp Trp Asp Lys 980 985 990 Asp Phe 1422 DNA Artificial Sequence PKG 14 acnggncanc cngaagcaag ca 22 15 26 DNAArtificial Sequence PKG 15 gcnaagagaa cttgnctgag ttcttc 26 16 28 DNAArtificial Sequence PKG 16 gaaggactac ncaaggcnga aggactgc 28

What is claimed is:
 1. A cGMP dependent protein kinase of a protozoan.2. A cGMP dependent protein kinase of a protozoan of the familyApicomplexan.
 3. A cGMP dependent protein kinase of an Eimeria species.4. A cGMP dependent protein kinase of Einieria tenellla.
 5. A cGMPdependent protein kinase of Toxoplasma gondii.
 6. A polynucleotideencoding a cGMP dependent protein kinase of SEQ ID NO:
 11. 7. Apolynucleotide having SEQ ID NO:
 10. 8. A polypeptide having SEQ ID NO:11.
 10. A polynucleotide encoding a cGMP dependent protein kinase of SEQID NO:
 13. 7. A polynucleotide having SEQ ID NO:
 12. 8. A polypeptidehaving SEQ ID NO:
 13. 9. A method for identifying compounds havingantiprotozoal activity comprising: (a) contacting protozoal PKG with (i)a known amount of a labeled compound that interacts with a PKG and (ii)a known dilution of a test compound or a natural product extract; and(b) quantitating the percent inhibition of interaction of said labeledcompound induced by said test compound.
 10. A method of claim 9 whereinsaid compound that interacts with PKG is a substrate for PKG.
 11. Amethod of claim 9 wherein said compound that interacts with PKG is acompound that binds to the enzyme.
 12. method for identifying compoundshaving antiprotozoal activity comprises: (a) contacting an intact hostor protozoal cell with a test compound or a natural product extract; (b)disrupting said cell to obtain a biochemical fraction possessing PKGcatalytic activity; and (c) determining the level of PKG activity insaid biochemical fraction.